Polypeptide variants with altered effector function

ABSTRACT

The present invention concerns polypeptides comprising a variant Fc region. More particularly, the present invention concerns Fc region-containing polypeptides that have altered effector function as a consequence of one or more amino acid modifications in the Fc region thereof.

[0001] This is a divisional application of non-provisional applicationSer. No. 09/483,588 which claims priority under 35 USC § 119 toprovisional application No. 60/116,023 filed Jan. 15, 1999, the entiredisclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention concerns polypeptides comprising a variantFc region. More particularly, the present invention concerns Fcregion-containing polypeptides that have altered effector function as aconsequence of one or more amino acid modifications in the Fc regionthereof.

[0004] 2. Description of Related Art

[0005] Antibodies are proteins which exhibit binding specificity to aspecific antigen. Native antibodies are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one end (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light and heavy chain variable domains.

[0006] The term “variable” refers to the fact that certain portions ofthe variable domains differ extensively in sequence among antibodies andare responsible for the binding specificity of each particular antibodyfor its particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called complementarity determiningregions (CDRs) both in the light chain and the heavy chain variabledomains. The more highly conserved portions of the variable domains arecalled the framework regions (FRs). The variable domains of native heavyand light chains each comprise four FRs, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FRs and, with the CDRsfrom the other chain, contribute to the formation of the antigen bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

[0007] The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions.Depending on the amino acid sequence of the constant region of theirheavy chains, antibodies or immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavychain constant regions that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. Of thevarious human immunoglobulin classes, only human IgG1, IgG2, IgG3 andIgM are known to activate complement; and human IgG1 and IgG3 mediateADCC more effectively than IgG2 and IgG4.

[0008] A schematic representation of the native IgG1 structure is shownin FIG. 1, where the various portions of the native antibody moleculeare indicated. Papain digestion of antibodies produces two identicalantigen binding fragments, called Fab fragments, each with a singleantigen binding site, and a residual “Fc” fragment, whose name reflectsits ability to crystallize readily. The crystal structure of the humanIgG Fc region has been determined (Deisenhofer, Biochemistry20:2361-2370 (1981)). In human IgG molecules, the Fc region is generatedby papain cleavage N-terminal to Cys 226. The Fc region is central tothe effector functions of antibodies.

[0009] The effector functions mediated by the antibody Fc region can bedivided into two categories: (1) effector functions that operate afterthe binding of antibody to an antigen (these functions involve theparticipation of the complement cascade or Fc receptor (FcR)-bearingcells); and (2) effector functions that operate independently of antigenbinding (these functions confer persistence in the circulation and theability to be transferred across cellular barriers by transcytosis).Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995).

[0010] While binding of an antibody to the requisite antigen has aneutralizing effect that might prevent the binding of a foreign antigento its endogenous target (e.g. receptor or ligand), binding alone maynot remove the foreign antigen. To be efficient in removing and/ordestructing foreign antigens, an antibody should be endowed with bothhigh affinity binding to its antigen, and efficient effector functions.

[0011] Fc Receptor (FcR) Binding

[0012] The interaction of antibodies and antibody-antigen complexes withcells of the immune system effects a variety of responses, includingantibody-dependent cell-mediated cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC) (reviewed in Daëron, Annu. Rev. Immunol.15:203-234 (1997); Ward and Ghetie, Therapeutic Immunol. 2:77-94 (1995);as well as Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991)).

[0013] Several antibody effector functions are mediated by Fc receptors(FcRs), which bind the Fc region of an antibody. FcRs are defined bytheir specificity for immunoglobulin isotypes; Fc receptors for IgGantibodies are referred to as FcγR, for IgE as FcεR, for IgA as FcαR andso on. Three subclasses of FcγR have been identified: FcγRI (CD64),FcγRII (CD32) and FcγRIII (CD16). Because each FcγR subclass is encodedby two or three genes, and alternative RNA spicing leads to multipletranscripts, a broad diversity in FcγR isoforms exists. The three genesencoding the FcγRI subclass (FcγRIA, FcγRIB and FcγRIC) are clustered inregion 1q21.1 of the long arm of chromosome 1; the genes encoding FcγRIIisoforms (FcγRIIA, FcγRIIB and FcγRIIC) and the two genes encodingFcγRIII (FcγRIIIA and FcγRIIIB) are all clustered in region 1q22. Thesedifferent FcR subtypes are expressed on different cell types (reviewedin Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492(1991)). For example,in humans, FcγRIIIB is found only on neutrophils, whereas FcγRIIIA isfound on macrophages, monocytes, natural killer (NK) cells, and asubpopulation of T-cells. Notably, FcγRIIIA is the only FcR present onNK cells, one of the cell types implicated in ADCC.

[0014] FcγRI, FcγRII and FcγRIII are immunoglobulin superfamily (IgSF)receptors; FcγRI has three IgSF domains in its extracellular domain,while FcγRII and FcγRII have only two IgSF domains in theirextracellular domains.

[0015] Another type of Fc receptor is the neonatal Fc receptor (FcRn).FcRn is structurally similar to major histocompatibility complex (MHC)and consists of an α-chain noncovalently bound to β2-microglobulin.

[0016] The binding site on human and murine antibodies for FcγR havebeen previously mapped to the so-called “lower hinge region” consistingof residues 233-239 (EU index numbering as in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Woof et al. Molec.Immunol. 23:319-330 (1986); Duncan et al. Nature 332:563 (1988);Canfield and Morrison, J. Exp. Med. 173:1483-1491 (1991); Chappel etal., Proc. Natl. Acad. Sci USA 88:9036-9040 (1991). Of residues 233-239,P238 and S239 have been cited as possibly being involved in binding, butthese two residues have never been evaluated by substitution ordeletion.

[0017] Other previously cited areas possibly involved in binding to FcγRare: G316-K338 (human IgG) for human FcγRI (by sequence comparison only;no substitution mutants were evaluated) (Woof et al. Molec. Immunol.23:319-330 (1986)); K274-R301 (human IgG1) for human FcγRIII (based onpeptides) (Sarmay et al. Molec. Immunol. 21:43-51 (1984)); Y407-R416(human IgG) for human FcγRIII (based on peptides) (Gergely et al.Biochem. Soc. Trans. 12:739-743 (1984)); as well as N297 and E318(murine IgG2b) for murine FcγRII (Lund et al., Molec. Immunol., 29:53-59(1992)).

[0018] Pro331 in IgG3 was changed to Ser, and the affinity of thisvariant to target cells analyzed. The affinity was found to be six-foldlower than that of unmutated IgG3, indicating the involvement of Pro331in FcγRI binding. Morrison et al., Immunologist, 2:119-124 (1994); andCanfield and Morrison, J. Exp. Med. 173:1483-91 (1991).

[0019] C1q Binding

[0020] C1q and two serine proteases, C1r and C1s, form the complex C1,the first component of the complement dependent cytotoxicity (CDC)pathway. C1q is a hexavalent molecule with a molecular weight ofapproximately 460,000 and a structure likened to a bouquet of tulips inwhich six collagenous “stalks” are connected to six globular headregions. Burton and Woof, Advances in Immunol. 51:1-84 (1992). Toactivate the complement cascade, it is necessary for C1q to bind to atleast two molecules of IgG1, IgG2, or IgG3 (the consensus is that IgG4does not activate complement), but only one molecule of IgM, attached tothe antigenic target. Ward and Ghetie, Therapeutic Immunology 2:77-94(1995) at page 80.

[0021] Based upon the results of chemical modifications andcrystallographic studies, Burton et al. (Nature, 288:338-344 (1980))proposed that the binding site for the complement subcomponent C1q onIgG involves the last two (C-terminal) β-strands of the CH2 domain.Burton later suggested (Molec. Immunol., 22(3):161-206 (1985)) that theregion comprising amino acid residues 318 to 337 might be involved incomplement fixation.

[0022] Duncan and Winter (Nature 332:738-40 (1988)), using site directedmutagenesis, reported that Glu318, Lys320 and Lys322 form the bindingsite to C1q. The data of Duncan and Winter were generated by testing thebinding of a mouse IgG2b isotype to guinea pig C1q. The role of Glu318,Lys320 and Lys322 residues in the binding of C1q was confirmed by theability of a short synthetic peptide containing these residues toinhibit complement mediated lysis. Similar results are disclosed in U.S.Pat. No. 5,648,260 issued on Jul. 15, 1997, and U.S. Pat. No. 5,624,821issued on Apr. 29, 1997.

[0023] The residue Pro331 has been implicated in C1q binding by analysisof the ability of human IgG subclasses to carry out complement mediatedcell lysis. Mutation of Ser331 to Pro331 in IgG4 conferred the abilityto activate complement. (Tao et al., J. Exp. Med., 178:661 -667 (1993);Brekke et al., Eur. J. Immunol., 24:2542-47 (1994)).

[0024] From the comparison of the data of the Winter group, and the Taoet al. and Brekke et al. papers, Ward and Ghetie concluded in theirreview article that there are at least two different regions involved inthe binding of C1q: one on the β-strand of the CH2 domain bearing theGlu318, Lys320 and Lys322 residues, and the other on a turn located inclose proximity to the same β-strand, and containing a key amino acidresidue at position 331.

[0025] Other reports suggested that human IgG1residues Leu235, andGly237, located in the lower hinge region, play a critical role incomplement fixation and activation. Xu et al., Journal of Immunology150:152A (Abstract) (1993). WO94/29351 published Dec. 22, 1994 reportsthat amino acid residues necessary for C1q and FcR binding of human IgG1are located in the N-terminal region of the CH2 domain, i.e. residues231 to 238.

[0026] It has further been proposed that the ability of IgG to bind C1qand activate the complement cascade also depends on the presence,absence, or modification of the carbohydrate moiety positioned betweenthe two CH2 domains (which is normally anchored at Asn297). Ward andGhetie, Therapeutic Immunology 2:77-94 (1995) at page 81.

SUMMARY OF THE INVENTION

[0027] The present invention provides a variant of a parent polypeptidecomprising an Fc region, which variant mediates antibody-dependentcell-mediated cytotoxicity (ADCC) in the presence of human effectorcells more effectively, or binds an Fc gamma receptor (FcγR) with betteraffinity, than the parent polypeptide and comprises at least one aminoacid modification in the Fc region. The polypeptide variant may, forexample, comprise an antibody or an immunoadhesin. The Fc region of theparent polypeptide preferably comprises a human Fc region; e.g., a humanIgG1, IgG2, IgG3 or IgG4 Fc region. The polypeptide variant preferablycomprises an amino acid modification (e.g. a substitution) at any one ormore of amino acid positions 256, 290, 298, 312, 326, 330, 333, 334,360, 378 or 430 of the Fc region, wherein the numbering of the residuesin the Fc region is that of the EU index as in Kabat.

[0028] In addition, the invention provides a polypeptide comprising avariant Fc region with altered Fc gamma receptor (FcγR) bindingaffinity, which polypeptide comprises an amino acid modification at anyone or more of amino acid positions 238, 239, 248, 249, 252, 254, 255,256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286,289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312,315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338,340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434,435, 437, 438 or 439 of the Fc region, wherein the numbering of theresidues in the Fc region is that of the EU index as in Kabat. Thevariant Fc region preferably comprises a variant human IgG Fc region,e.g., a variant human IgG1, IgG2, IgG3 or IgG4 Fc region. In thisrespect, it is noted that, in the work in the above-cited art where theparent polypeptide had a non-human murine Fc region, different residuesfrom those identified herein were thought to impact FcR binding. Forexample, in the murine IgG2b/murine FcγRII system, IgG E318 was found tobe important for binding (Lund et al. Molec. Immunol. 27(1):53-59(1992)), whereas E318A had no effect in the human IgG/human FcγRIIsystem (Table 6 below).

[0029] In one embodiment, the polypeptide variant with altered FcγRbinding activity displays reduced binding to an FcγR and comprises anamino acid modification at any one or more of amino acid positions 238,239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293,294, 295, 296, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340,373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438 or 439 of theFc region, wherein the numbering of the residues in the Fc region isthat of the EU index as in Kabat.

[0030] For example, the polypeptide variant may display reduced bindingto an FcγRI and comprise an amino acid modification at any one or moreof amino acid positions 238, 265, 269, 270, 327 or 329 of the Fc region,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat.

[0031] The polypeptide variant may display reduced binding to an FcγRIIand comprise an amino acid modification at any one or more of amino acidpositions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329,333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 of the Fcregion, wherein the numbering of the residues in the Fc region is thatof the EU index as in Kabat.

[0032] The polypeptide variant of interest may display reduced bindingto an FcγRIII and comprise an amino acid modification at one or more ofamino acid positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270,272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338, 340,373,376, 382, 388, 389, 416, 434, 435 or 437 of the Fc region, whereinthe numbering of the residues in the Fc region is that of the EU indexas in Kabat.

[0033] In another embodiment, the polypeptide variant with altered FcγRbinding affinity displays improved binding to the FcγR and comprises anamino acid modification at any one or more of amino acid positions 255,256, 258, 267, 268, 272, 276, 280, 283, 285, 286, 290, 298, 301, 305,307, 309,312, 315, 320, 322, 326, 330, 331, 333, 334, 337, 340, 360,378, 398 or 430 of the Fc region, wherein the numbering of the residuesin the Fc region is that of the EU index as in Kabat.

[0034] For example, the polypeptide variant may display increasedbinding to an FcγRIII and, optionally, may further display decreasedbinding to an FcγRII. An exemplary such variant comprises amino acidmodification(s) at position(s) 298 and/or 333 of the Fc region, whereinthe numbering of the residues in the Fc region is that of the EU indexas in Kabat.

[0035] The polypeptide variant may display increased binding to anFcγRII and comprise an amino acid modification at any one or more ofamino acid positions 255, 256, 258, 267, 268, 272, 276, 280, 283, 285,286, 290, 301, 305, 307, 309, 312, 315, 320, 322, 326, 330, 331, 337,340, 378, 398 or 430 of the Fc region, wherein the numbering of theresidues in the Fc region is that of the EU index as in Kabat. Suchpolypeptide variants with increased binding to an FcγRII may optionallyfurther display decreased binding to an FcγRIII and may, for example,comprise an amino acid modification at any one or more of amino acidpositions 268, 272, 298, 301, 322 or 340 of the Fc region, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat.

[0036] The invention further provides a polypeptide comprising a variantFc region with altered neonatal Fc receptor (FcRn) binding affinity,which polypeptide comprises an amino acid modification at any one ormore of amino acid positions 238, 252, 253, 254, 255, 256, 265, 272,286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376,378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 or447 of the Fc region, wherein the numbering of the residues in the Fcregion is that of the EU index as in Kabat. Such polypeptide variantswith reduced binding to an FcRn may comprise an amino acid modificationat any one or more of amino acid positions 252, 253, 254, 255, 288, 309,386, 388, 400, 415, 433, 435, 436, 439 or 447 of the Fc region, whereinthe numbering of the residues in the Fc region is that of the EU indexas in Kabat. The above-mentioned polypeptide variants may,alternatively, display increased binding to FcRn and comprise an aminoacid modification at any one or more of amino acid positions 238, 256,265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376,378, 380, 382, 413,424 or 434 of the Fc region, wherein the numbering ofthe residues in the Fc region is that of the EU index as in Kabat.

[0037] The invention also provides a composition comprising thepolypeptide variant and a physiologically or pharmaceutically acceptablecarrier or diluent. This composition for potential therapeutic use issterile and may be lyophilized.

[0038] Diagnostic and therapeutic uses for the polypeptide variantsdisclosed herein are contemplated. In one diagnostic application, theinvention provides a method for determining the presence of an antigenof interest comprising exposing a sample suspected of containing theantigen to the polypeptide variant and determining binding of thepolypeptide variant to the sample. In one therapeutic application, theinvention provides a method of treating a mammal suffering from orpredisposed to a disease or disorder, comprising administering to themammal a therapeutically effective amount of a polypeptide variant asdisclosed herein, or of a composition comprising the polypeptide variantand a pharmaceutically acceptable carrier.

[0039] The invention further provides: isolated nucleic acid encodingthe polypeptide variant; a vector comprising the nucleic acid,optionally, operably linked to control sequences recognized by a hostcell transformed with the vector; a host cell containing the vector; amethod for producing the polypeptide variant comprising culturing thishost cell so that the nucleic acid is expressed and, optionally,recovering the polypeptide variant from the host cell culture (e.g. fromthe host cell culture medium).

[0040] The invention further provides a method for making a variant Fcregion with altered Fc receptor (FcR) binding affinity, or alteredantibody-dependent cell-mediated cytotoxicity (ADCC) activity,comprising:

[0041] (a) introducing one or more amino acid modifications into an Fcregion of a parent polypeptide in order to generate a variant Fc region;

[0042] (b) determining binding of the variant Fc region to an FcR, ordetermining ADCC activity of the variant Fc region.

[0043] Step (b) of the method may comprise determining binding of thevariant Fc region to one or more FcRs in vitro. Moreover, the method mayresult in the identification of a variant Fc region with improved FcRbinding affinity, or with improved ADCC activity, in step (b) thereof.Where step (b) comprises determining binding of the Fc region to an FcR,the FcR may, for example, be human Fc gamma receptor III (FcγRIII).Where step (b) comprises determining binding of the variant Fc region toat least two different FcRs, the FcRs tested preferably include human Fcgamma receptor II (FcγRII) and human Fc gamma receptor III (FcγRIII).

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 is a schematic representation of a native IgG. Disulfidebonds are represented by heavy lines between CH1 and CL domains and thetwo CH2 domains. V is variable domain; C is constant domain; L standsfor light chain and H stands for heavy chain.

[0045]FIG. 2 shows C1q binding of wild type (wt) C2B8 antibody; C2B8antibody with a human IgG2 constant region (IgG2); and variants K322A,K320A and E318A.

[0046]FIG. 3 depicts C1q binding of variants P331A, P329A and K322A.

[0047]FIGS. 4A and 4B depict the amino acid sequences of E27 anti-IgEantibody light chain (FIG. 4A; SEQ ID NO:1) and heavy chain (FIG. 4B;SEQ ID NO:2).

[0048]FIG. 5 is a schematic diagram of the “immune complex” prepared foruse in the FcR assay described in Example 1. The hexamer comprisingthree anti-IgE antibody molecules (the “Fc region-containingpolypeptide”) and three IgE molecules (the “first target molecule”) isshown. IgE has two “binding sites” for the anti-IgE antibody (E27) inthe Fc region thereof. Each IgE molecule in the complex is further ableto bind two VEGF molecules (“the second target polypeptide”). VEGF hastwo “binding sites” for IgE.

[0049]FIG. 6 shows C1q binding results obtained for variants D270K andD270V compared to wild type C2B8.

[0050]FIG. 7 depicts complement dependent cytotoxicity (CDC) of variantsD270K and D270V, compared to wild type C2B8.

[0051]FIG. 8 shows C1q binding ELISA results for 293 cell-produced wildtype C2B8 antibody (293-Wt-C2B8), CHO-produced wild type C2B8 antibody(CHO-Wt-C2B8) and various variant antibodies.

[0052]FIG. 9 shows C1q binding ELISA results obtained for wild type (wt)C2B8 and various variant antibodies as determined in Example 3.

[0053]FIG. 10 depicts the three-dimensional structure of a human IgG Fcregion, highlighting residues: Asp270, Lys326, Pro329, Pro331, Lys322and Glu333.

[0054]FIG. 11 shows C1q binding ELISA results obtained for wild typeC2B8 and various variant antibodies as determined in Example 3.

[0055]FIG. 12 shows C1q binding ELISA results obtained for wild typeC2B8 and double variants, K326M-E333S and K326A-E333A.

[0056]FIG. 13 shows CDC of wild type C2B8 and double variants,K326M-E333S and K326A-E333A.

[0057]FIG. 14 depicts C1q binding ELISA results obtained for C2B8 with ahuman IgG4 (IgG4), wild type C2B8 (Wt-C2B8), C2B8 with a human IgG2constant region (IgG2), and variant antibodies as described in Example3.

[0058]FIGS. 15A and 15B show binding patterns for parent antibody (E27)to FcγRIIB and FcγRIIIA. FIG. 15A shows the binding pattern for thehumanized anti-IgE E27 IgG1 as a monomer (open circles), hexamer (closedsquares), and immune complex consisting of multiple hexamers (closedtriangles) to a recombinant GST fusion protein of the human FcγRIIB(CD32) receptor α subunit. The hexameric complex (closed squares) wasformed by the mixture of equal molar concentrations of E27 (which bindsto the Fc region of human IgE) and a human myeloma IgE. The hexamer is astable 1.1 kD complex consisting of 3 IgG molecules (150 kD each) and 3IgE molecules (200 kD each). The immune complex (closed triangles) wasformed sequentially by first mixing equal molar concentrations of E27and recombinant anti-VEGF IgE (human IgE with Fab variable domains thatbind human VEGF) to form the hexamer. Hexamers were then linked to forman immune complex by the addition of 2×molar concentration of humanVEGF, a 44 kD homodimer which has two binding sites for the anti-VEGFIgE per mole of VEGF. FIG. 15B shows the binding pattern to arecombinant GST fusion protein of the human FcγRIIIA (CD16) receptor αsubunit.

[0059]FIG. 16A shows the binding of immune complexes using differentantigen-antibody pairs to recombinant GST fusion protein of the FcγRIIAreceptor α subunit. FIG. 16B shows the binding of the sameantigen-antibody pairs to the GST fusion protein of the FcγRIIIAreceptor α subunit. Closed circles represent binding of humanIgE:anti-IgE E27 IgG1; open circles represent binding of humanVEGF:humanized anti-VEGF IgG1.

[0060]FIG. 17 summarizes differences in binding selectivity of somealanine variants between the different FcγRs. Binding of alaninevariants at residues in the CH2 domain of anti-IgE E27 IgG1 are shown toFcγRIIA, FcγRIIB, and FcγRIIIA. Type 1 abrogates binding to all threereceptors: D278A (265 in EU numbering). Type 2 improves binding toFcγRIIA and FcγRIIB, while binding to FcγRIIIA is unaffected: S280A (267in EU numbering). Type 3 improves binding to FcγRIIA and FcγRIIB, butreduces binding to FcγRIIIA: H281A (268 in EU numbering). Type 4 reducesbinding to FcγRIIA and FcγRIIB, while improving binding to FcγRIIIA:S317A (298 in EU numbering). Type 5 improves binding to FcγRIIIA, butdoes not affect binding to FcγRIIA and FcγRIIB: E352A, K353A (333 and334 in EU numbering).

[0061]FIGS. 18A and 18B compare the FcγRIIIA protein/protein assay andCHO GPI-FcγRIIIA cell based assay, respectively. FIG. 18A illustratesbinding of selected alanine variants to FcγRIIIA-GST fusion protein.S317A (298 in EU numbering) and S317A/K353A (298 and 334 in EUnumbering) bind better than E27 wildtype, while D278A (265 in EUnumbering) almost completely abrogates binding. FIG. 18B illustratesthat a similar pattern of binding is found on CHO cells expressing arecombinant GPI-linked form of FcγRIIIA.

[0062]FIGS. 19A and 19B compare the FcγRIIB protein/protein assay andCHO GPI-FcγRIIB cell based assay, respectively. FIG. 19A illustratesbinding of selected alanine variants to FcγRIIB-GST fusion protein.H281A (268 in EU numbering) binds better than E27 wildtype while S317A(298 in EU numbering) shows reduced binding. FIG. 19B illustrates that asimilar pattern of binding is found on CHO cells expressing arecombinant membrane bound form of FcγRIIB.

[0063]FIG. 20 shows single alanine substitutions in the CH2 domain ofanti-HER2 IgG1(HERCEPTIN®) that influence FcγRIIIA binding in both theprotein-protein and cell-based assays alter the ability to bind toFcγRIIIA on peripheral blood mononuclear cell (PBMC) effector cells.Recombinant humanized anti-HER2 (HERCEPTIN®), which binds toHER2-expressing SK-BR-3 breast tumor cells, was preincubated with⁵¹Cr-labeled SK-BR-3 cells for 30 minutes (opsonization) at 100 ng/ml(filled circles) and 1.25 ng/ml (filled squares). Keeping the SK-BR-3tumor target cell concentration constant, the ratio of effector cellswas increased from 0 to 100. The spontaneous cytotoxicity in the absenceof antibody (hatched squares) was 20% at an effector:target (E:T) ratioof 100:1. A single alanine mutation that did not affect FcγRIIIAbinding, variant G31=R309A (292 in EU numbering), did not effect ADCC(filled triangles). A single alanine mutation that only slightlyincreased binding to FcγRIIIA, variant G30=K307A (290 in EU numbering),also showed slightly improved ADCC (i.e., a 1.1 fold improvement in ADCCactivity, calculated as area under the curve) at 1.25 ng/ml at all E:Tratios (filled diamonds) compared to wildtype antibody at 1.25 ng/ml(filled square). A single alanine mutation that decreased binding toFcγRIIIA, variant G34=Q312A (295 in EU numbering), also showed decreasedADCC activity (filled inverted triangles).

[0064]FIG. 21 illustrates that a single alanine mutation which had themost improved binding to FcγRIIIA, variant G36=S317A (298 in EUnumbering), in the protein-protein and cell-based assays also showed themost improvement in ADCC (filled triangles) among the variants comparedto wildtype (closed squares) at 1.25 ng/ml. G36 displayed a 1.7 foldimprovement in ADCC activity, calculated as area under the curve.Variants G17=E282A (269 in EU numbering) and G18=D283A (270 in EUnumbering) both showed reduced binding to FcγRIIIA as well as reducedefficacy in ADCC. The effector cells were PBMCs.

[0065]FIG. 22A depicts alignments of native sequence IgG Fc regions.Native sequence human IgG Fc region sequences, humIgG1(non-A and Aallotypes) (SEQ ID NOs: 3 and 4, respectively), humIgG2 (SEQ ID NO:5),humIgG3 (SEQ ID NO:6) and humIgG4 (SEQ ID NO:7), are shown. The humanIgG1 sequence is the non-A allotype, and differences between thissequence and the A allotype (at positions 356 and 358; EU numberingsystem) are shown below the human IgG1 sequence. Native sequence murineIgG Fc region sequences, murIgG1 (SEQ ID NO:8), murIgG2A (SEQ ID NO:9),murIgG2B (SEQ ID NO:10) and murIgG3 (SEQ ID NO:11), are also shown. FIG.22B shows percent identity among the Fc region sequences of FIG. 22A.

[0066]FIG. 23 depicts alignments of native sequence human IgG Fc regionsequences, humIgG1 (non-A and A allotypes; SEQ ID NOs:3 and 4,respectively), humIgG2 (SEQ ID NO:5), humIgG3 (SEQ ID NO:6) and humIgG4(SEQ ID NO:7) with differences between the sequences marked withasterisks.

[0067]FIG. 24 shows area under curve (AUC) for selected variantscompared to anti-HER2 IgG1 (HERCEPTIN®) in a 4 hour ADCC assay. Theeffector cells were PBMCs (N=5). Variant G36 (S317A; 298 in Eunumbering) with improved binding to FcγRIIIA showed improved ADCCactivity; variant G31 (R309A; 292 in Eu numbering) which did not displayaltered FcγRIIIA binding, also had unaltered ADCC activity; and G14(D265A; 278 in Eu numbering) which had reduced FcγRIIIA binding, alsohad reduced ADCC activity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] I. Definitions

[0069] Throughout the present specification and claims, the numbering ofthe residues in an immunoglobulin heavy chain is that of the EU index asin Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991), expressly incorporated herein by reference. The “EU index as inKabat” refers to the residue numbering of the human IgG1EU antibody.

[0070] A “parent polypeptide” is a polypeptide comprising an amino acidsequence which lacks one or more of the Fc region modificationsdisclosed herein and which differs in effector function compared to apolypeptide variant as herein disclosed. The parent polypeptide maycomprise a native sequence Fc region or an Fc region with pre-existingamino acid sequence modifications (such as additions, deletions and/orsubstitutions).

[0071] The term “Fc region” is used to define a C-terminal region of animmunoglobulin heavy chain, e.g., as shown in FIG. 1. The “Fc region”may be a native sequence Fc region or a variant Fc region. Although theboundaries of the Fc region of an immunoglobulin heavy chain might vary,the human IgG heavy chain Fc region is usually defined to stretch froman amino acid residue at position Cys226, or from Pro230, to thecarboxyl-terminus thereof. The Fc region of an immunoglobulin generallycomprises two constant domains, CH2 and CH3, as shown, for example, inFIG. 1.

[0072] The “CH2 domain” of a human IgG Fc region (also referred to as“Cγ2” domain) usually extends from about amino acid 231 to about aminoacid 340. The CH2 domain is unique in that it is not closely paired withanother domain. Rather, two N-linked branched carbohydrate chains areinterposed between the two CH2 domains of an intact native IgG molecule.It has been speculated that the carbohydrate may provide a substitutefor the domain-domain pairing and help stabilize the CH2 domain. Burton,Molec. Immunol.22:161-206 (1985).

[0073] The “CH3 domain” comprises the stretch of residues C-terminal toa CH2 domain in an Fc region (i.e. from about amino acid residue 341 toabout amino acid residue 447 of an IgG)

[0074] A “functional Fc region” possesses an “effector function” of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays as herein disclosed, for example.

[0075] A “native sequence Fc region” comprises an amino acid sequenceidentical to the amino acid sequence of an Fc region found in nature.Native sequence human Fc regions are shown in FIG. 23 and include anative sequence human IgG1Fc region (non-A and A allotypes); nativesequence human IgG2 Fc region; native sequence human IgG3 Fc region; andnative sequence human IgG4 Fc region as well as naturally occurringvariants thereof. Native sequence murine Fc regions are shown in FIG.22A.

[0076] A “variant Fc region” comprises an amino acid sequence whichdiffers from that of a native sequence Fc region by virtue of at leastone “amino acid modification” as herein defined. Preferably, the variantFc region has at least one amino acid substitution compared to a nativesequence Fc region or to the Fc region of a parent polypeptide, e.g.from about one to about ten amino acid substitutions, and preferablyfrom about one to about five amino acid substitutions in a nativesequence Fc region or in the Fc region of the parent polypeptide. Thevariant Fc region herein will preferably possess at least about 80%homology with a native sequence Fc region and/or with an Fc region of aparent polypeptide, and most preferably at least about 90% homologytherewith, more preferably at least about 95% homology therewith.

[0077] “Homology” is defined as the percentage of residues in the aminoacid sequence variant that are identical after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percenthomology. Methods and computer programs for the alignment are well knownin the art. One such computer program is “Align 2”, authored byGenentech, Inc., which was filed with user documentation in the UnitedStates Copyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

[0078] The term “Fc region-containing polypeptide” refers to apolypeptide, such as an antibody or immunoadhesin (see definitionsbelow), which comprises an Fc region.

[0079] The terms “Fc receptor” or “FcR” are used to describe a receptorthat binds to the Fc region of an antibody. The preferred FcR is anative sequence human FcR. Moreover, a preferred FcR is one which bindsan IgG antibody (a gamma receptor) and includes receptors of the FcγRI,FcγRII, and FcγRIII subclasses, including allelic variants andalternatively spliced forms of these receptors. FcγRII receptors includeFcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibitingreceptor”), which have similar amino acid sequences that differprimarily in the cytoplasmic domains thereof. Activating receptorFcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB containsan immunoreceptor tyrosine-based inhibition motif (ITIM) in itscytoplasmic domain. (see review M. in Daëron, Annu. Rev. Immunol.15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); andde Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)).

[0080] “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” referto a cell-mediated reaction in which nonspecific cytotoxic cells thatexpress FcRs (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol9:457-92 (1991).

[0081] “Human effector cells” are leukocytes which express one or moreFcRs and perform effector functions. Preferably, the cells express atleast FcγRIII and perform ADCC effector function. Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils; with PBMCs and NK cells being preferred. The effector cellsmay be isolated from a native source thereof, e.g. from blood or PBMCsas described herein.

[0082] A polypeptide variant with “altered” FcR binding affinity or ADCCactivity is one which has either enhanced or diminished FcR bindingactivity and/or ADCC activity compared to a parent polypeptide or to apolypeptide comprising a native sequence Fc region. The polypeptidevariant which “displays increased binding” to an FcR binds at least oneFcR with better affinity than the parent polypeptide. The polypeptidevariant which “displays decreased binding” to an FcR, binds at least oneFcR with worse affinity than a parent polypeptide. Such variants whichdisplay decreased binding to an FcR may possess little or no appreciablebinding to an FcR, e.g., 0-20% binding to the FcR compared to a nativesequence IgG Fc region, e.g. as determined in the Examples herein.

[0083] The polypeptide variant which binds an FcR with “better affinity”than a parent polypeptide, is one which binds any one or more of theabove identified FcRs with substantially better binding affinity thanthe parent antibody, when the amounts of polypeptide variant and parentpolypeptide in the binding assay are essentially the same. For example,the polypeptide variant with improved FcR binding affinity may displayfrom about 1.15 fold to about 100 fold, e.g. from about 1.2 fold toabout 50 fold improvement in FcR binding affinity compared to the parentpolypeptide, where FcR binding affinity is determined, for example, asdisclosed in the Examples herein.

[0084] The polypeptide variant which “mediates antibody-dependentcell-mediated cytotoxicity (ADCC) in the presence of human effectorcells more effectively” than a parent antibody is one which in vitro orin vivo is substantially more effective at mediating ADCC, when theamounts of polypeptide variant and parent antibody used in the assay areessentially the same. Generally, such variants will be identified usingthe in vitro ADCC assay as herein disclosed, but other assays or methodsfor determining ADCC activity, e.g. in an animal model etc, arecontemplated. The preferred variant is from about 1.5 fold to about 100fold, e.g. from about two fold to about fifty fold, more effective atmediating ADCC than the parent, e.g. in the in vitro assay disclosedherein.

[0085] An “amino acid modification” refers to a change in the amino acidsequence of a predetermined amino acid sequence. Exemplary modificationsinclude an amino acid substitution, insertion and/or deletion. Thepreferred amino acid modification herein is a substitution.

[0086] An “amino acid modification at” a specified position, e.g. of theFc region, refers to the substitution or deletion of the specifiedresidue, or the insertion of at least one amino acid residue adjacentthe specified residue. By insertion “adjacent” a specified residue ismeant insertion within one to two residues thereof. The insertion may beN-terminal or C-terminal to the specified residue.

[0087] An “amino acid substitution” refers to the replacement of atleast one existing amino acid residue in a predetermined amino acidsequence with another different “replacement” amino acid residue. Thereplacement residue or residues may be “naturally occurring amino acidresidues” (i.e. encoded by the genetic code) and selected from the groupconsisting of: alanine (Ala); arginine (Arg); asparagine (Asn); asparticacid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu);glycine (Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine(Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine(Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine(Val). Preferably, the replacement residue is not cysteine. Substitutionwith one or more non-naturally occurring amino acid residues is alsoencompassed by the definition of an amino acid substitution herein. A“non-naturally occurring amino acid residue” refers to a residue, otherthan those naturally occurring amino acid residues listed above, whichis able to covalently bind adjacent amino acid residues(s) in apolypeptide chain. Examples of non-naturally occurring amino acidresidues include norleucine, ornithine, norvaline, homoserine and otheramino acid residue analogues such as those described in Ellman et al.Meth. Enzym. 202:301-336 (1991). To generate such non-naturallyoccurring amino acid residues, the procedures of Noren et al. Science244:182 (1989) and Ellman et al., supra, can be used. Briefly, theseprocedures involve chemically activating a suppressor tRNA with anon-naturally occurring amino acid residue followed by in vitrotranscription and translation of the RNA.

[0088] An “amino acid insertion” refers to the incorporation of at leastone amino acid into a predetermined amino acid sequence. While theinsertion will usually consist of the insertion of one or two amino acidresidues, the present application contemplates larger “peptideinsertions”, e.g. insertion of about three to about five or even up toabout ten amino acid residues. The inserted residue(s) may be naturallyoccurring or non-naturally occurring as disclosed above.

[0089] An “amino acid deletion” refers to the removal of at least oneamino acid residue from a predetermined amino acid sequence.

[0090] “Hinge region” is generally defined as stretching from Glu2l6 toPro230 of human IgG1 (Burton, Molec. Immunol.22:161-206 (1985)). Hingeregions of other IgG isotypes may be aligned with the IgG1 sequence byplacing the first and last cysteine residues forming inter-heavy chainS-S bonds in the same positions.

[0091] The “lower hinge region” of an Fc region is normally defined asthe stretch of residues immediately C-terminal to the hinge region, i.e.residues 233 to 239 of the Fc region. Prior to the present invention,FcγR binding was generally attributed to amino acid residues in thelower hinge region of an IgG Fc region.

[0092] “C1q” is a polypeptide that includes a binding site for the Fcregion of an immunoglobulin. C1q together with two serine proteases, C1rand C1s, forms the complex C1, the first component of the complementdependent cytotoxicity (CDC) pathway. Human C1q can be purchasedcommercially from, e.g. Quidel, San Diego, Calif.

[0093] The term “binding domain” refers to the region of a polypeptidethat binds to another molecule. In the case of an FcR, the bindingdomain can comprise a portion of a polypeptide chain thereof (e.g. the αchain thereof) which is responsible for binding an Fc region. One usefulbinding domain is the extracellular domain of an FcR α chain.

[0094] The term “antibody” is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments so long as theyexhibit the desired biological activity.

[0095] “Antibody fragments”, as defined for the purpose of the presentinvention, comprise a portion of an intact antibody, generally includingthe antigen binding or variable region of the intact antibody or the Fcregion of an antibody which retains FcR binding capability. Examples ofantibody fragments include linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.The antibody fragments preferably retain at least part of the hinge andoptionally the CH1 region of an IgG heavy chain. More preferably, theantibody fragments retain the entire constant region of an IgG heavychain, and include an IgG light chain.

[0096] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations that typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler et al., Nature 256:495 (1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al., Nature 352:624-628 (1991) andMarks et al., J. Mol. Biol. 222:581-597 (1991), for example.

[0097] The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[0098] “Humanized” forms of non-human (e.g., murine) antibodies arechimeric antibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525(1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0099] The term “hypervariable region” when used herein refers to theamino acid residues of an antibody which are responsible forantigen-binding. The hypervariable region comprises amino acid residuesfrom a “complementarity determining region” or “CDR” (i.e. residues24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domainand 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain; Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991)) and/or those residues from a “hypervariable loop” (i.e.residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917(1987)). “Framework” or “FR” residues are those variable domain residuesother than the hypervariable region residues as herein defined.

[0100] As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the “binding domain” of a heterologous “adhesin”protein (e.g. a receptor, ligand or enzyme) with an immunoglobulinconstant domain. Structurally, the immunoadhesins comprise a fusion ofthe adhesin amino acid sequence with the desired binding specificitywhich is other than the antigen recognition and binding site (antigencombining site) of an antibody (i.e. is “heterologous”) and animmunoglobulin constant domain sequence.

[0101] The term “ligand binding domain” as used herein refers to anynative cell-surface receptor or any region or derivative thereofretaining at least a qualitative ligand binding ability of acorresponding native receptor. In a specific embodiment, the receptor isfrom a cell-surface polypeptide having an extracellular domain that ishomologous to a member of the immunoglobulin supergenefamily. Otherreceptors, which are not members of the immunoglobulin supergenefamilybut are nonetheless specifically covered by this definition, arereceptors for cytokines, and in particular receptors with tyrosinekinase activity (receptor tyrosine kinases), members of thehematopoietin and nerve growth factor receptor superfamilies, and celladhesion molecules, e.g. (E-, L- and P-) selectins.

[0102] The term “receptor binding domain” is used to designate anynative ligand for a receptor, including cell adhesion molecules, or anyregion or derivative of such native ligand retaining at least aqualitative receptor binding ability of a corresponding native ligand.This definition, among others, specifically includes binding sequencesfrom ligands for the above-mentioned receptors.

[0103] An “antibody-immunoadhesin chimera” comprises a molecule thatcombines at least one binding domain of an antibody (as herein defined)with at least one immunoadhesin (as defined in this application).Exemplary antibody-immunoadhesin chimeras are the bispecific CD4-IgGchimeras described in Berg et al., PNAS (USA) 88:4723-4727 (1991) andChamow et a., J. Immunol. 153:4268 (1994).

[0104] An “isolated” polypeptide is one that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain. Isolated polypeptide includes the polypeptide in situ withinrecombinant cells since at least one component of the polypeptide'snatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

[0105] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures. Those in need of treatment include thosealready with the disorder as well as those in which the disorder is tobe prevented.

[0106] A “disorder” is any condition that would benefit from treatmentwith the polypeptide variant. This includes chronic and acute disordersor diseases including those pathological conditions which predispose themammal to the disorder in question. In one embodiment, the disorder iscancer.

[0107] The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidneycancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma and various types of head and neck cancer.

[0108] A “HER2-expressing cancer” is one comprising cells which haveHER2 receptor protein (Semba et al., PNAS (USA) 82:6497-6501 (1985) andYamamoto et al. Nature 319:230-234 (1986) (Genebank accession numberX03363)) present at their cell surface, such that an anti-HER2 antibodyis able to bind to the cancer.

[0109] The word “label” when used herein refers to a detectable compoundor composition which is conjugated directly or indirectly to thepolypeptide. The label may be itself be detectable (e.g., radioisotopelabels or fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

[0110] An “isolated” nucleic acid molecule is a nucleic acid moleculethat is identified and separated from at least one contaminant nucleicacid molecule with which it is ordinarily associated in the naturalsource of the polypeptide nucleic acid. An isolated nucleic acidmolecule is other than in the form or setting in which it is found innature. Isolated nucleic acid molecules therefore are distinguished fromthe nucleic acid molecule as it exists in natural cells. However, anisolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily express the polypeptide where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

[0111] The expression “control sequences” refers to DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, and a ribosome binding site. Eukaryotic cells are known toutilize promoters, polyadenylation signals, and enhancers.

[0112] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0113] As used herein, the expressions “cell,” “cell line,” and “cellculture” are used interchangeably and all such designations includeprogeny. Thus, the words “transformants” and “transformed cells” includethe primary subject cell and cultures derived therefrom without regardfor the number of transfers. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included. Where distinct designations are intended, it will be clearfrom the context.

[0114] The term “molecular complex” when used herein refers to therelatively stable structure which forms when two or more heterologousmolecules (e.g. polypeptides) bind (preferably noncovalently) to oneanother. The preferred molecular complex herein is an immune complex.

[0115] “Immune complex” refers to the relatively stable structure whichforms when at least one target molecule and at least one heterologous Fcregion-containing polypeptide bind to one another forming a largermolecular weight complex. Examples of immune complexes areantigen-antibody aggregates and target molecule-immunoadhesinaggregates. The term “immune complex” as used herein, unless indicatedotherwise, refers to an ex vivo complex (i.e. other than the form orsetting in which it may be found in nature). However, the immune complexmay be administered to a mammal, e.g. to evaluate clearance of theimmune complex in the mammal.

[0116] The term “target molecule” refers to a molecule, usually apolypeptide, which is capable of being bound by a heterologous moleculeand has one or more binding sites for the heterologous molecule. Theterm “binding site” refers to a region of a molecule to which anothermolecule can bind. The “first target molecule” herein comprises at leasttwo distinct binding sites (for example, two to five separate bindingsites) for an analyte (e.g. an Fc region-containing polypeptide) suchthat at least two analyte molecules can bind to the first targetmolecule. In the preferred embodiment of the invention, the two or morebinding sites are identical (e.g. having the same amino acid sequence,where the target molecule is a polypeptide). In Example 1 below, thefirst target molecule was IgE and had two separate binding sites in theFc region thereof to which the Fc region-containing polypeptide (ananti-IgE antibody, E27) could bind. Other first target molecules includedimers of substantially identical monomors (e.g. neurotrophins, IL8 andVEGF) or are polypeptides comprising two or more substantially identicalpolypeptide chains (e.g. antibodies or immunoadhesins). The “secondtarget molecule” comprises at least two distinct binding sites (forexample, two to five separate binding sites) for the first targetmolecule such that at least two first target molecules can bind to thesecond target molecule. Preferably, the two or more binding sites areidentical (e.g. having the same amino acid sequence, where the targetmolecule is a polypeptide). In Example 2, the second target molecule wasVEGF, which has a pair of distinct binding sites to which the variabledomain of the IgE antibody could bind. Other second target molecules arecontemplated, e.g. other dimers of substantially identical monomers(e.g. neurotrophins or IL8) or polypeptides comprising two or moresubstantially identical domains (e.g. antibodies or immunoadhesins).

[0117] An “analyte” is a substance that is to be analyzed. The preferredanalyte is an Fc region-containing polypeptide that is to be analyzedfor its ability to bind to an Fc receptor.

[0118] A “receptor” is a polypeptide capable of binding at least oneligand. The preferred receptor is a cell-surface receptor having anextracellular ligand-binding domain and, optionally, other domains (e.g.transmembrane domain, intracellular domain and/or membrane anchor). Thereceptor to be evaluated in the assay described herein may be an intactreceptor or a fragment or derivative thereof (e.g. a fusion proteincomprising the binding domain of the receptor fused to one or moreheterologous polypeptides). Moreover, the receptor to be evaluated forits binding properties may be present in a cell or isolated andoptionally coated on an assay plate or some other solid phase.

[0119] The phrase “low affinity receptor” denotes a receptor that has aweak binding affinity for a ligand of interest, e.g. having a bindingconstant of about 50 nM or worse affinity. Exemplary low affinityreceptors include FcγRII and FcγRIII.

[0120] II. Modes for Carrying Out the Invention

[0121] The invention herein relates to a method for making a polypeptidevariant. The “parent”, “starting” or “nonvariant” polypeptide isprepared using techniques available in the art for generatingpolypeptides comprising an Fc region. In the preferred embodiment of theinvention, the parent polypeptide is an antibody and exemplary methodsfor generating antibodies are described in more detail in the followingsections. The parent polypeptide may, however, be any other polypeptidecomprising an Fc region, e.g. an immunoadhesin. Methods for makingimmunoadhesins are elaborated in more detail hereinbelow.

[0122] In an alternative embodiment, a variant Fc region may begenerated according to the methods herein disclosed and this “variant Fcregion” can be fused to a heterologous polypeptide of choice, such as anantibody variable domain or binding domain of a receptor or ligand.

[0123] The parent polypeptide comprises an Fc region. Generally the Fcregion of the parent polypeptide will comprise a native sequence Fcregion, and preferably a human native sequence Fc region. However, theFc region of the parent polypeptide may have one or more pre-existingamino acid sequence alterations or modifications from a native sequenceFc region. For example, the C1q binding activity of the Fc region mayhave been previously altered (other types of Fc region modifications aredescribed in more detail below). In a further embodiment the parentpolypeptide Fc region is “conceptual” and, while it does not physicallyexist, the antibody engineer may decide upon a desired variant Fc regionamino acid sequence and generate a polypeptide comprising that sequenceor a DNA encoding the desired variant Fc region amino acid sequence.

[0124] In the preferred embodiment of the invention, however, a nucleicacid encoding an Fc region of a parent polypeptide is available and thisnucleic acid sequence is altered to generate a variant nucleic acidsequence encoding the Fc region variant.

[0125] DNA encoding an amino acid sequence variant of the startingpolypeptide is prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, preparation by site-directed(or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared DNA encoding the polypeptide

[0126] Site-directed mutagenesis is a preferred method for preparingsubstitution variants. This technique is well known in the art (see,e.g.,Carter et al. Nucleic Acids Res. 13:4431-4443 (1985) and Kunkel etal., Proc. Natl. Acad. Sci. USA 82:488 (1985)). Briefly, in carryingoutsite-directed mutagenesis of DNA, the starting DNA is altered byfirst hybridizing an oligonucleotide encoding the desired mutation to asingle strand of such starting DNA. After hybridization, a DNApolymerase is used to synthesize an entire second strand, using thehybridized oligonucleotide as a primer, and using the single strand ofthe starting DNA as a template. Thus, the oligonucleotide encoding thedesired mutation is incorporated in the resulting double-stranded DNA.

[0127] PCR mutagenesis is also suitable for making amino acid sequencevariants of the starting polypeptide. See Higuchi, in PCR Protocols,pp.177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acids Res.17:723-733 (1989). Briefly, when small amounts of template DNA are usedas starting material in a PCR, primers that differ slightly in sequencefrom the corresponding region in a template DNA can be used to generaterelatively large quantities of a specific DNA fragment that differs fromthe template sequence only at the positions where the primers differfrom the template.

[0128] Another method for preparing variants, cassette mutagenesis, isbased on the technique described by Wells et al., Gene 34:315-323(1985). The starting material is the plasmid (or other vector)comprising the starting polypeptide DNA to be mutated. The codon(s) inthe starting DNA to be mutated are identified. There must be a uniquerestriction endonuclease site on each side of the identified mutationsite(s). If no such restriction sites exist, they may be generated usingthe above-described oligonucleotide-mediated mutagenesis method tointroduce them at appropriate locations in the starting polypeptide DNA.The plasmid DNA is cut at these sites to linearize it. A double-strandedoligonucleotide encoding the sequence of the DNA between the restrictionsites but containing the desired mutation(s) is synthesized usingstandard procedures, wherein the two strands of the oligonucleotide aresynthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 5′ and 3′ ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedDNA sequence.

[0129] Alternatively, or additionally, the desired amino acid sequenceencoding a polypeptide variant can be determined, and a nucleic acidsequence encoding such amino acid sequence variant can be generatedsynthetically.

[0130] The amino acid sequence of the parent polypeptide is modified inorder to generate a variant Fc region with altered Fc receptor bindingaffinity or activity in vitro and/or in vivo and/or alteredantibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitroand/or in vivo.

[0131] Generally, the modification entails one or more amino acidsubstitutions. In one embodiment, the replacement residue does notcorrespond to a residue present in the same position in any of thenative sequence Fc regions in FIG. 22A. For example, according to thisembodiment of the invention, Pro331 of a human IgG3 or IgG1 Fc region isreplaced with a residue other than Ser (the corresponding alignedresidue found in native sequence human IgG4). In one embodiment, theresidue in the parent polypeptide which is substituted with areplacement residue is not an alanine and/or is not residue Ala339 of anFc region. In the case of an amino acid substitution, preferably theresidue in the parent polypeptide is replaced with an alanine residue.However, the present invention contemplates replacement of the residueof the parent polypeptide with any other amino acid residue. Thesubstitution may, for example, be a “conservative substitution”. Suchconservative substitutions are shown in Table 1 under the heading of“preferred substitution”. More substantial changes may be achieved bymaking one or more “exemplary substitutions” which are not the preferredsubstitution in Table 1. TABLE 1 Original Exemplary Preferred ResidueSubstitutions Substitution Ala (A) val; leu; ile val Arg (R) lys; gln;asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser serGln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln;lys; arg arg Ile (I) leu; val; met; ala; leu phe; norleucine Leu (L)norleucine; ile; val; ile met; ala; phe Lys (K) arg; gln; asn arg Met(M) leu; phe; ile leu Phe (F) leu; val; ile; ala; leu tyr Pro (P) alaala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp;phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala; norleucine

[0132] Substantial modifications in the biological properties of the Fcregion may be accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

[0133] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0134] (2) neutral hydrophilic: cys, ser, thr;

[0135] (3) acidic: asp, glu;

[0136] (4) basic: asn, gin, his, lys, arg;

[0137] (5) residues that influence chain orientation: gly, pro; and

[0138] (6) aromatic: trp, tyr, phe.

[0139] Non-conservative substitutions will entail exchanging a member ofone of these classes for a member of another class. Conservative andnon-conservative amino acid substitutions are exemplified in Table 8hereinbelow.

[0140] As is demonstrated in Example 4 herein, one can engineer an Fcregion variant with altered binding affinity for one or more FcRs. Aswas shown in that Example, different classes of Fc region variants canbe made e.g,. as summarized in the following table. Where the variant Fcregion has more than one amino acid substitution, generally, but notnecessarily, amino acid substitutions in the same class are combined toachieve the desired result. TABLE 2 CLASSES OF Fc REGION VARIANTS ClassFcR binding property Position of Fc region substitution(s) 1A reducedbinding to all FcγR 238, 265, 269, 270, 297*, 327, 329 1B reducedbinding to both FcγRII and 239, 294, 295, 303, 338, 373, 376, 416, 435FcγRIII 2 improved binding to both FcγRII and 256, 290, 312, 326, 330,339^(#), 378, 430 FcγRIII 3 improved binding to FcγRII and no 255, 258,267, 276, 280, 283, 285, 286, 305, effect on FcγRIII binding 307, 309,315, 320, 331, 337, 398 4 improved binding to FcγRII and 268, 272, 301,322, 340 reduced binding to FcγRIII 5 reduced binding to FcγRII and no292, 324, 335, 414, 419, 438, 439 effect on FcγRIII binding 6 reducedbinding to FcγRII and 298, 333 improved binding to FcγRIII 7 no effecton FcγRII binding and 248, 249, 252, 254, 278, 289, 293, 296, 338,reduced binding to FcγRIII 382, 388, 389, 434, 437 8 no effect on FcγRIIbinding and 334, 360 improved binding to FcγRIII

[0141] Aside from amino acid substitutions, the present inventioncontemplates other modifications of the parent Fc region amino acidsequence in order to generate an Fc region variant with altered effectorfunction.

[0142] One may, for example, delete one or more amino acid residues ofthe Fc region in order reduce binding to an FcR. Generally, one willdelete one or more of the Fc region residues identified herein aseffecting FcR binding (see Example 4 below) in order to generate such anFc ion variant. Generally, no more than one to about ten Fc regionresidues will be deleted according to this embodiment of the invention.The Fc region herein comprising one or more amino acid deletions willpreferably retain at least about 80%, and preferably at least about 90%,d most preferably at least about 95%, of the parent Fc region or of anative sequence human region.

[0143] One may also make amino acid insertion Fc region variants, whichvariants have altered effector function. For example, one may introduceat least one amino acid residue (e.g. one to o amino acid residues andgenerally no more than ten residues) adjacent to one or more of theregion positions identified herein as impacting FcR binding. By“adjacent” is meant within one two amino acid residues of a Fc regionresidue identified herein. Such Fc region variants may play enhanced ordiminished FcR binding and/or ADCC activity. In order to generate suchinsertion variants, one may evaluate a co-crystal structure of apolypeptide comprising a binding region of an FcR (e.g. theextracellular domain of the FcR of interest) and the Fc region intowhich the amino acid residue(s) are to be inserted (see, for example,Deisenhofer, Biochemistry 20(9):2361-2370 (1981); and Burmeister et al.,Nature 372:379-383, (1994)) in order to rationally design an Fc regionvariant with, e.g., improved FcR binding ability. Such insertion(s) willgenerally be made in an Fc region loop, but not in the secondarystructure (i.e. in a β-strand) of the Fc region.

[0144] By introducing the appropriate amino acid sequence modificationsin a parent Fc region, one can generate a variant Fc region which (a)mediates antibody-dependent cell-mediated cytotoxicity (ADCC) in thepresence of human effector cells more effectively and/or (b) binds an Fcgamma receptor (FcγR) with better affinity than the parent polypeptide.Such Fc region variants will generally comprise at least one amino acidmodification in the Fc region. Combining amino acid modifications isthought to be particularly desirable. For example, the variant Fc regionmay include two, three, four, five, etc substitutions therein, e.g. ofthe specific Fc region positions identified herein.

[0145] Preferably, the parent polypeptide Fc region is a human Fcregion, e.g. a native sequence human Fc region human IgG1 (A and non-Aallotypes), IgG2, IgG3 or IgG4 Fc region. Such sequences are shown inFIG. 23.

[0146] To generate an Fc region with improved ADCC activity, the parentpolypeptide preferably has pre-existing ADCC activity, e.g., itcomprises a human IgG1 or human IgG3 Fc region. In one embodiment, thevariant with improved ADCC mediates ADCC substantially more effectivelythan an antibody with a native sequence IgG1 or IgG3 Fc region and theantigen-binding region of the variant. Preferably, the variantcomprises, or consists essentially of, substitutions of two or three ofthe residues at positions 298, 333 and 334 of the Fc region. Mostpreferably, residues at positions 298, 333 and 334 are substituted,(e.g. with alanine residues). Moreover, in order to generate the Fcregion variant with improved ADCC activity, one will generally engineeran Fc region variant with improved binding affinity for FcγRIII, whichis thought to be an important FcR for mediating ADCC. For example, onemay introduce an amino acid modification (e.g. a substitution) into theparent Fc region at any one or more of amino acid positions 256, 290,298, 312, 326, 330, 333, 334, 360, 378 or 430 to generate such avariant. The variant with improved binding affinity for FcγRIII mayfurther have reduced binding affinity for FcγRII, especially reducedaffinity for the inhibiting FcγRIIB receptor.

[0147] The amino acid modification(s) are preferably introduced into theCH2 domain of a Fc region, since the experiments herein indicate thatthe CH2 domain is important for FcR binding activity. Moreover, unlikethe teachings of the above-cited art, the instant applicationcontemplates the introduction of a modification into a part of the Fcregion other than in the lower hinge region thereof.

[0148] Useful amino acid positions for modification in order to generatea variant IgG Fc region with altered Fc gamma receptor (FcγR) bindingaffinity or activity include any one or more of amino acid positions238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270,272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296,298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329,330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388,389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fcregion. Preferably, the parent Fc region used as the template togenerate such variants comprises a human IgG Fc region. Where residue331 is substituted, the parent Fc region is preferably not human nativesequence IgG3, or the variant Fc region comprising a substitution atposition 331 preferably displays increased FcR binding, e.g. to FcγRII.

[0149] To generate an Fc region variant with reduced binding to the FcγRone may introduce an amino acid modification at any one or more of aminoacid positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272,278, 289, 292, 293, 294, 295, 296, 298, 301, 303, 322, 324, 327, 329,333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435,437, 438 or 439 of the Fc region.

[0150] Variants which display reduced binding to FcγRI, include thosecomprising an Fc region amino acid modification at any one or more ofamino acid positions 238, 265, 269, 270, 327 or 329.

[0151] Variants which display reduced binding to FcγRII include thosecomprising an Fc region amino acid modification at any one or more ofamino acid positions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324,327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439.

[0152] Fc region variants which display reduced binding to FcγRIIIinclude those comprising an Fc region amino acid modification at any oneor more of amino acid positions 238, 239, 248, 249, 252, 254, 265, 268,269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329,338, 340, 373, 376, 382, 388, 389, 416, 434, 435 or 437.

[0153] Variants with improved binding to one or more FcγRs may also bemade. Such Fc region variants may comprise an amino acid modification atany one or more of amino acid positions 255, 256, 258, 267, 268, 272,276, 280, 283, 285, 286, 290, 298, 301, 305, 307, 309, 312, 315, 320,322, 326, 330, 331, 333, 334, 337, 340, 360, 378, 398 or 430 of the Fcregion.

[0154] For example, the variant with improved FcγR binding activity maydisplay increased binding to FcγRIII, and optionally may further displaydecreased binding to FcγRII; e.g. the variant may comprise an amino acidmodification at position 298 and/or 333 of an Fc region.

[0155] Variants with increased binding to FcγRII include thosecomprising an amino acid modification at any one or more of amino acidpositions 255, 256, 258, 267, 268, 272, 276, 280, 283, 285, 286, 290,301, 305, 307, 309, 312, 315, 320, 322, 326, 330, 331, 337, 340, 378,398 or 430 of an Fc region. Such variants may further display decreasedbinding to FcγRII. For example, they may include an Fc region amino acidmodification at any one or more of amino acid positions 268, 272, 298,301, 322 or 340.

[0156] While it is preferred to alter binding to a FcγR, Fc regionvariants with altered binding affinity for the neonatal receptor (FcRn)are also contemplated herein. Fc region variants with improved affinityfor FcRn are anticipated to have longer serum half-lives, and suchmolecules will have useful applications in methods of treating mammalswhere long half-life of the administered polypeptide is desired, e.g.,to treat a chronic disease or disorder. Fc region variants withdecreased FcRn binding affinity, on the contrary, are expected to haveshorter half-lives, and such molecules may, for example, be administeredto a mammal where a shortened circulation time may be advantageous, e.g.for in vivo diagnostic imaging or for polypeptides which have toxic sideeffects when left circulating in the blood stream for extended periods,etc. Fc region variants with decreased FcRn binding affinity areanticipated to be less likely to cross the placenta, and thus may beutilized in the treatment of diseases or disorders in pregnant women.

[0157] Fc region variants with altered binding affinity for FcRn includethose comprising an Fc region amino acid modification at any one or moreof amino acid positions 238, 252, 253, 254, 255, 256, 265, 272, 286,288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378,380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 or 447.Those which display reduced binding to FcRn will generally comprise anFc region amino acid modification at any one or more of amino acidpositions 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435,436, 439 or 447; and those with increased binding to FcRn will usuallycomprise an Fc region amino acid modification at any one or more ofamino acid positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312,317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434.

[0158] The polypeptide variant(s) prepared as described above may besubjected to further modifications, oftentimes depending on the intendeduse of the polypeptide. Such modifications may involve furtheralteration of the amino acid sequence (substitution, insertion and/ordeletion of amino acid residues), fusion to heterologous polypeptide(s)and/or covalent modifications. Such “further modifications” may be madeprior to, simultaneously with, or following, the amino acidmodification(s) disclosed above which result in an alteration of Fcreceptor binding and/or ADCC activity. In one embodiment, one maycombine the Fc region modification herein with Fc region substitutionsdisclosed in the references cited in the “Related Art” section of thisapplication.

[0159] Alternatively or additionally, it may be useful to combine theabove amino acid modifications with one or more further amino acidmodifications that alter C1q binding and/or complement dependentcytoxicity function of the Fc region.

[0160] The starting polypeptide of particular interest herein is usuallyone that binds to C1q and displays complement dependent cytotoxicity(CDC). The further amino acid substitutions described herein willgenerally serve to alter the ability of the starting polypeptide to bindto C1q and/or modify its complement dependent cytotoxicity function,e.g. to reduce and preferably abolish these effector functions. However,polypeptides comprising substitutions at one or more of the describedpositions with improved C1q binding and/or complement dependentcytotoxicity (CDC) function are contemplated herein. For example, thestarting polypeptide may be unable to bind C1q and/or mediate CDC andmay be modified according to the teachings herein such that it acquiresthese further effector functions. Moreover, polypeptides withpre-existing C1q binding activity, optionally further having the abilityto mediate CDC may be modified such that one or both of these activitiesare enhanced.

[0161] To generate an Fc region with altered C1q binding and/orcomplement dependent cytotoxicity (CDC) function, the amino acidpositions to be modified are generally selected from heavy chainpositions 270, 322, 326, 327, 329, 331, 333, and 334, where thenumbering of the residues in an IgG heavy chain is that of the EU indexas in Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991). In one embodiment, only one of the eight above-identifiedpositions is altered in order to generate the polypeptide variant regionwith altered C1q binding and/or complement dependent cytotoxicity (CDC)function. Preferably only residue 270, 329 or 322 is altered if this isthe case. Alternatively, two or more of the above-identified positionsare modified. If substitutions are to be combined, generallysubstitutions which enhance human C1q binding (e.g. at residue positions326, 327, 333 and 334) or those which diminish human C1q binding (e.g.,at residue positions 270, 322, 329 and 331) are combined. In the latterembodiment, all four positions (i.e., 270, 322, 329 and 331) may besubstituted. Preferably, further substitutions at two, three or all ofpositions 326, 327, 333 or 334 are combined, optionally with other Fcregion substitutions, to generate a polypeptide with improved human C1qbinding and preferably improved CDC activity in vitro or in vivo.

[0162] Proline is conserved at position 329 in human IgG's. This residueis preferably replaced with alanine, however substitution with any otheramino acid is contemplated, e.g., serine, threonine, asparagine, glycineor valine.

[0163] Proline is conserved at position 331 in human IgG1, IgG2 andIgG3, but not IgG4 (which has a serine residue at position 331). Residue331 is preferably replaced by alanine or another amino acid, e.g. serine(for IgG regions other than IgG4), glycine or valine.

[0164] Lysine 322 is conserved in human IgGs, and this residue ispreferably replaced by an alanine residue, but substitution with anyother amino acid residue is contemplated, e.g. serine, threonine,glycine or valine.

[0165] D270 is conserved in human IgGs, and this residue may be replacedby another amino acid residue, e.g. alanine, serine, threonine, glycine,valine, or lysine.

[0166] K326 is also conserved in human IgGs. This residue may besubstituted with another residue including, but not limited to, valine,glutamic acid, alanine, glycine, aspartic acid, methionine ortryptophan, with tryptophan being preferred.

[0167] Likewise, E333 is also conserved in human IgGs. E333 ispreferably replaced by an amino acid residue with a smaller side chainvolume, such as valine, glycine, alanine or serine, with serine beingpreferred.

[0168] K334 is conserved in human IgGs and may be substituted withanother residue such as alanine or other residue.

[0169] In human IgG1 and IgG3, residue 327 is an alanine. In order togenerate a variant with improved C1q binding, this alanine may besubstituted with another residue such as glycine. In IgG2 and IgG4,residue 327 is a glycine and this may be replaced by alanine (or anotherresidue) to diminish C1q binding.

[0170] As disclosed above, one can design an Fc region with alteredeffector function, e.g., by modifying C1q binding and/or FcR binding andthereby changing CDC activity and/or ADCC activity. For example, one cangenerate a variant Fc region with improved C1q binding and improvedFcγRIII binding; e.g. having both improved ADCC activity and improvedCDC activity. Alternatively, where one desires that effector function bereduced or ablated, one may engineer a variant Fc region with reducedCDC activity and/or reduced ADCC activity. In other embodiments, one mayincrease only one of these activities, and optionally also reduce theother activity, e.g. to generate an Fc region variant with improved ADCCactivity, but reduced CDC activity and vice versa.

[0171] With respect to further amino acid sequence alterations, anycysteine residue not involved in maintaining the proper conformation ofthe polypeptide variant also may be substituted, generally with serine,to improve the oxidative stability of the molecule and prevent aberrantcross linking.

[0172] Another type of amino acid substitution serves to alter theglycosylation pattern of the polypeptide. This may be achieved bydeleting one or more carbohydrate moieties found in the polypeptide,and/or adding one or more glycosylation sites that are not present inthe polypeptide. Glycosylation of polypeptides is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Addition of glycosylation sites to thepolypeptide is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original polypeptide(for O-linked glycosylation sites). An exemplary glycosylation varianthas an amino acid substitution of residue Asn 297 of the heavy chain.

[0173] Moreover, the class, subclass or allotype of the Fc region may bealtered by one or more further amino acid substitutions to generate anFc region with an amino acid sequence more homologous to a differentclass, subclass or allotype as desired. For example, a murine Fc regionmay be altered to generate an amino acid sequence more homologous to ahuman Fc region; a human non-A allotype IgG1 Fc region may be modifiedto achieve a human A allotype IgG1 Fc region etc. In one embodiment, theamino modification(s) herein which alter FcR binding and/or ADCCactivity are made in the CH2 domain of the Fc region and the CH3 domainis deleted or replaced with another dimerization domain. Preferably,however, the CH3 domain is retained (aside from amino acid modificationstherein which alter effector function as herein disclosed).

[0174] The polypeptide variant may be subjected to one or more assays toevaluate any change in biological activity compared to the startingpolypeptide.

[0175] Preferably the polypeptide variant essentially retains theability to bind antigen compared to the nonvariant polypeptide, i.e. thebinding capability is no worse than about 20 fold, e.g. no worse thanabout 5 fold of that of the nonvariant polypeptide. The bindingcapability of the polypeptide variant may be determined using techniquessuch as fluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA), for example.

[0176] The ability of the polypeptide variant to bind an FcR may beevaluated. Where the FcR is a high affinity Fc receptor, such as FcγRI,FcRn or FcγRIIIA-V158, binding can be measured by titrating monomericpolypeptide variant and measuring bound polypeptide variant using anantibody which specifically binds to the polypeptide variant in astandard ELISA format (see Example 2 below). Another FcR binding assayfor low affinity FcRs is described in Examples 1 and 4.

[0177] To assess ADCC activity of the polypeptide variant, an in vitroADCC assay, such as that described in Example 4 may be performed usingvarying effector:target ratios. Useful “effector cells” for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of thepolypeptide variant may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0178] The ability of the variant to bind C1q and mediate complementdependent cytotoxicity (CDC) may be assessed.

[0179] To determine C1q binding, a C1q binding ELISA may be performed.Briefly, assay plates may be coated overnight at 4°C. with polypeptidevariant or starting polypeptide (control) in coating buffer. The platesmay then be washed and blocked. Following washing, an aliquot of humanC1q may be added to each well and incubated for 2 hrs at roomtemperature. Following a further wash, 100 μl of a sheep anti-complementC1q peroxidase conjugated antibody may be added to each well andincubated for 1 hour at room temperature. The plate may again be washedwith wash buffer and 100 μl of substrate buffer containing OPD(O-phenylenediamine dihydrochloride (Sigma)) may be added to each well.The oxidation reaction, observed by the appearance of a yellow color,may be allowed to proceed for 30 minutes and stopped by the addition of100 μl of 4.5 N H₂SO₄. The absorbance may then read at (492-405) nm.

[0180] An exemplary polypeptide variant is one that displays a“significant reduction in C1q binding” in this assay. This means thatabout 100 μg/ml of the polypeptide variant displays about fold or morereduction in C1q binding compared to 100μg/ml of a control antibodyhaving a nonmutated IgG1 Fc region. In the most preferred embodiment,the polypeptide variant“does not bind C1q”, i.e. 100 μg/ml of thepolypeptide variant displays about 100 fold or more reduction in C1qbinding compared to 100 μg/ml of the control antibody.

[0181] Another exemplary variant is one which “has a better bindingaffinity for human C1q than the parent polypeptide”. Such a molecule maydisplay, for example, about two-fold or more, and preferably aboutfive-fold or more, improvement in human C1q binding compared to theparent polypeptide (e.g. at the IC₅₀ values for these two molecules).For example, human C1q binding may be about two-fold to about 500-fold,and preferably from about two-fold or from about five-fold to about1000-fold improved compared to the parent polypeptide.

[0182] To assess complement activation, a complement dependentcytotoxicity (CDC) assay may be performed, e.g. as described inGazzano-Santoro et al., J. Immunol. Methods 202:163 (1997). Briefly,various concentrations of the polypeptide variant and human complementmay be diluted with buffer. Cells which express the antigen to which thepolypeptide variant binds may be diluted to a density of ˜1×10⁶ cells/ml. Mixtures of polypeptide variant, diluted human complement and cellsexpressing the antigen may be added to a flat bottom tissue culture 96well plate and allowed to incubate for 2 hrs at 37° C. and 5% CO₂ tofacilitate complement mediated cell lysis. 50 μl of alamar blue (AccumedInternational) may then be added to each well and incubated overnight at37° C. The absorbance is measured using a 96-well fluorometer withexcitation at 530 nm and emission at 590 nm. The results may beexpressed in relative fluorescence units (RFU). The sampleconcentrations may be computed from a standard curve and the percentactivity as compared to nonvariant polypeptide is reported for thepolypeptide variant of interest.

[0183] Yet another exemplary variant “does not activate complement”. Forexample, 0.6 μg/ml of the polypeptide variant displays about 0-10% CDCactivity in this assay compared to a 0.6 μg/ml of a control antibodyhaving a nonmutated IgG1 Fc region. Preferably the variant does notappear to have any CDC activity in the above CDC assay.

[0184] The invention also pertains to a polypeptide variant withenhanced CDC compared to a parent polypeptide, e.g., displaying abouttwo-fold to about 100-fold improvement in CDC activity in vitro or invivo (e.g. at the IC₅₀ values for each molecule being compared).

[0185] A. Receptor Binding Assay and Immune Complex

[0186] A receptor binding assay has been developed herein which isparticularly useful for determining binding of an analyte of interest toa receptor where the affinity of the analyte for the receptor isrelatively weak, e.g. in the micromolar range as is the case forFcγRIIA, FcγRIIB, FcγRIIIA and FcγRIIIB. The method involves theformation of a molecular complex that has an improved avidity for thereceptor of interest compared to the noncomplexed analyte. The preferredmolecular complex is an immune complex comprising: (a) an Fcregion-containing polypeptide (such as an antibody or an immunoadhesin);(b) a first target molecule which comprises at least two binding sitesfor the Fc region-containing polypeptide; and (c) a second targetmolecule which comprises at least two binding sites for the first targetmolecule.

[0187] In Example 1 below, the Fc region-containing polypeptide is ananti-IgE antibody, such as the E27 antibody (FIGS. 4A-4B). E27, whenmixed with human IgE at an 1:1 molar ratio, forms a stable hexamerconsisting of three E27 molecules and three IgE molecules. In Example 1below, the “first target molecule” is a chimeric form of IgE in whichthe Fab portion of an anti-VEGF antibody is fused to the human IgE Fcportion and the “second target molecule” is the antigen to which the Fabbinds (i.e. VEGF). Each molecule of IgE binds two molecules of VEGF.VEGF also binds two molecules of IgE per molecule of VEGF. Whenrecombinant human VEGF was added at a 2:1 molar ratio to IgE:E27hexamers, the hexamers were linked into larger molecular weightcomplexes via the IgE:VEGF interaction (FIG. 5). The Fc region of theanti-IgE antibody of the resultant immune complex binds to FcR withhigher avidity than either uncomplexed anti-IgE or anti-IgE:IgEhexamers.

[0188] Other forms of molecular complexes for use in the receptor assayare contemplated. Examples comprising only an Fc region-containingpolypeptide:first target molecule combination include animmunoadhesin:ligand combination such as VEGF receptor(KDR)-immunoadhesin:VEGF and a full-length bispecific antibody(bsAb):first target molecule. A further example of an Fcregion-containing polypeptide:first target molecule:second targetmolecule combination include a nonblocking antibody:solublereceptor:ligand combination such as anti-Trk antibody:soluble Trkreceptor:neurotrophin (Urfer et al. J. Biol. Chem. 273(10):5829-5840(1998)).

[0189] Aside from use in a receptor binding assay, the immune complexesdescribed above have further uses including evaluation of Fcregion-containing polypeptide function and immune complex clearance invivo. Hence, the immune complex may be administered to a mammal (e.g. ina pre-clinical animal study) and evaluated for its half-life etc.

[0190] To determine receptor binding, a polypeptide comprising at leastthe binding domain of the receptor of interest (e.g. the extracellulardomain of an α subunit of an FcR) may be coated on solid phase, such asan assay plate. The binding domain of the receptor alone or areceptor-fusion protein may be coated on the plate using standardprocedures. Examples of receptor-fusion proteins includereceptor-glutathione S-transferase (GST) fusion protein, receptor-chitinbinding domain fusion protein, receptor-hexaHis tag fusion protein(coated on glutathione, chitin, and nickel coated plates, respectively).Alternatively, a capture molecule may be coated on the assay plate andused to bind the receptor-fusion protein via the non-receptor portion ofthe fusion protein. Examples include anti-hexaHis F(ab′)₂ coated on theassay plate used to capture receptor-hexaHis tail fusion or anti-GSTantibody coated on the assay plate used to capture a receptor-GSTfusion. In other embodiments, binding to cells expressing at least thebinding domain of the receptor may be evaluated. The cells may benaturally occurring hematopoietic cells that express the FcR of interestor may be transformed with nucleic acid encoding the FcR or a bindingdomain thereof such that the binding domain is expressed at the surfaceof the cell to be tested.

[0191] The immune complex described hereinabove is added to thereceptor-coated plates and incubated for a sufficient period of timesuch that the analyte binds to the receptor. Plates may then be washedto remove unbound complexes, and binding of the analyte may be detectedaccording to known methods. For example, binding may be detected using areagent (e.g. an antibody or fragment thereof) which binds specificallyto the analyte, and which is optionally conjugated with a detectablelabel (detectable labels and methods for conjugating them topolypeptides are described below in the section entitled“Non-Therapeutic Uses for the Polypeptide Variant”).

[0192] As a matter of convenience, the reagents can be provided in anassay kit, i.e., a packaged combination of reagents, for combinationwith the analyte in assaying the ability of the analyte to bind to areceptor of interest. The components of the kit will generally beprovided in predetermined ratios. The kit may provide the first targetmolecule and/or the second target molecule, optionally complexedtogether. The kit may further include assay plates coated with thereceptor or a binding domain thereof (e.g. the extracellular domain ofthe α subunit of an FcR). Usually, other reagents, such as an antibodythat binds specifically to the analyte to be assayed, labeled directlyor indirectly with an enzymatic label, will also be provided in the kit.Where the detectable label is an enzyme, the kit will include substratesand cofactors required by the enzyme (e.g. a substrate precursor whichprovides the detectable chromophore or fluorophore). In addition, otheradditives may be included such as stabilizers, buffers (e.g. assayand/or wash lysis buffer) and the like. The relative amounts of thevarious reagents may be varied widely to provide for concentrations insolution of the reagents that substantially optimize the sensitivity ofthe assay. Particularly, the reagents may be provided as dry powders,usually lyophilized, including excipients that on dissolution willprovide a reagent solution having the appropriate concentration. The kitalso suitably includes instructions for carrying out the assay.

[0193] B. Antibody Preparation

[0194] In the preferred embodiment of the invention, the Fcregion-containing polypeptide which is modified according to theteachings herein is an antibody. Techniques for producing antibodiesfollow:

[0195] (i) Antigen Selection and Preparation

[0196] Where the polypeptide is an antibody, it is directed against anantigen of interest. Preferably, the antigen is a biologically importantpolypeptide and administration of the antibody to a mammal sufferingfrom a disease or disorder can result in a therapeutic benefit in thatmammal. However, antibodies directed against nonpolypeptide antigens(such as tumor-associated glycolipid antigens; see U.S. Pat. No.5,091,178) are also contemplated.

[0197] Where the antigen is a polypeptide, it may be a transmembranemolecule (e.g. receptor) or ligand such as a growth factor. Exemplaryantigens include molecules such as renin; a growth hormone, includinghuman growth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor (TF),and von Willebrands factor; anti-clotting factors such as Protein C;atrial natriuretic factor; lung surfactant; a plasminogen activator,such as urokinase or human urine or tissue-type plasminogen activator(t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, orTGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of any of the above-listed polypeptides.

[0198] Preferred molecular targets for antibodies encompassed by thepresent invention include CD proteins such as CD3, CD4, CD8, CD1 9, CD20and CD34; members of the ErbB receptor family such as the EGF receptor,HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1,Mac1, p150.95, VLA-4, ICAM-1, VCAM, α4/β7 integrin, and αv/β3 integrinincluding either α or β subunits thereof (e.g. anti-CD11 a, anti-CD18 oranti-CD11b antibodies); growth factors such as VEGF; tissue factor (TF);alpha interferon (α-IFN); an interleukin, such as IL-8; IgE; blood groupantigens; flk2/fit3 receptor; obesity (OB) receptor; mpl receptor;CTLA-4; protein C etc.

[0199] Soluble antigens or fragments thereof, optionally conjugated toother molecules, can be used as immunogens for generating antibodies.For transmembrane molecules, such as receptors, fragments of these (e.g.the extracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

[0200] (ii) Polyclonal Antibodies

[0201] Polyclonal antibodies are preferably raised in animals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of therelevant antigen and an adjuvant. It may be useful to conjugate therelevant antigen to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

[0202] Animals are immunized against the antigen, immunogenicconjugates, or derivatives by combining, e.g., 100 μg or 5 μg of theprotein or conjugate (for rabbits or mice, respectively) with 3 volumesof Freund's complete adjuvant and injecting the solution intradermallyat multiple sites. One month later the animals are boosted with 1/5 to1/10 the original amount of peptide or conjugate in Freund's completeadjuvant by subcutaneous injection at multiple sites. Seven to 14 dayslater the animals are bled and the serum is assayed for antibody titer.Animals are boosted until the titer plateaus. Preferably, the animal isboosted with the conjugate of the same antigen, but conjugated to adifferent protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are suitably used toenhance the immune response.

[0203] (iii) Monoclonal Antibodies

[0204] Monoclonal antibodies may be made using the hybridoma methodfirst described by Kohler et al., Nature, 256:495 (1975), or may be madeby recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0205] In the hybridoma method, a mouse or other appropriate hostanimal, such as a hamster or macaque monkey, is immunized as hereinabovedescribed to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein used forimmunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp.59-103 (AcademicPress, 1986)).

[0206] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

[0207] Preferred myeloma cells are those that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred myeloma cell lines are murine myelomalines, such as those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif. USA, and SP-2 or X63-Ag8-653 cells available from the AmericanType Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)).

[0208] Culture medium in which hybridoma cells are growing is assayedfor production of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

[0209] After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

[0210] The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0211] DNA encoding the monoclonal antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the monoclonal antibodies). The hybridomacells serve as a preferred source of such DNA. Once isolated, the DNAmay be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. Recombinant production of antibodies willbe described in more detail below.

[0212] In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783. (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

[0213] The DNA also may be modified, for example, by substituting thecoding sequence for human heavy- and light-chain constant domains inplace of the homologous murine sequences (U.S. Pat. No. 4,816,567;Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.

[0214] Typically such non-immunoglobulin polypeptides are substitutedfor the constant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

[0215] (iv) Humanized and Human Antibodies

[0216] A humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0217] The choice of human variable domains, both light and heavy, to beused in making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol Biol., 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immnol., 151:2623 (1993)).

[0218] It is further important that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to a preferredmethod, humanized antibodies are prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

[0219] Alternatively, it is now possible to produce transgenic animals(e.g., mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); and Duchosal et al. Nature 355:258(1992). Human antibodies can also be derived from phage-displaylibraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks etal., J. Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech14:309 (1996)).

[0220] (v) Multispecific Antibodies

[0221] Multispecific antibodies have binding specificities for at leasttwo different antigens. While such molecules normally will only bind twoantigens (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein. Examples of BsAbs include those with onearm directed against a tumor cell antigen and the other arm directedagainst a cytotoxic trigger molecule such as anti-FcγRI/anti-CD15,anti-p185^(HER2)/FcγRIII (CD16), anti-CD3/anti-malignant B-cell (1D10),anti-CD3/anti-p185^(HER2), anti-CD3/anti-p97, anti-CD3/anti-renal cellcarcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma),ant-CD3/anti-melanocyte stimulating hormone analog, anti-EGFreceptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19,anti-CD3/MoV18, anti-neural cell ahesion molecule (NCAM)/anti-CD3,anti-folate binding protein (FBP)/anti-CD3, anti-pan carcinomaassociated antigen (AMOC-31)/anti-CD3; BsAbs with one arm which bindsspecifically to a tumor antigen and one arm which binds to a toxin suchas anti-saporin/anti-Id-1, anti-CD22/anti-saporin,anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin Achain, anti-interferon-α (IFN-α)/anti-hybridoma idiotype,anti-CEA/anti-vinca alkaloid; BsAbs for converting enzyme activatedprodrugs such as anti-CD30/anti-alkaline phosphatase (which catalyzesconversion of mitomycin phosphate prodrug to mitomycin alcohol); BsAbswhich can be used as fibrinolytic agents such as anti-fibrin/anti-tissueplasminogen activator (tPA), anti-fibrin/anti-urokinase-type plasminogenactivator (uPA); BsAbs for targeting immune complexes to cell surfacereceptors such as anti-low density lipoprotein (LDL)/anti-Fc receptor(e.g. FcγRI, FcγRII or FcγRIII); BsAbs for use in therapy of infectiousdiseases such as anti-CD3/anti-herpes simplex virus (HSV), anti-T-cellreceptor:CD3 complex/anti-influenza, anti-FcγR/anti-HIV; BsAbs for tumordetection in vitro or in vivo such as anti-CEA/anti-EOTUBE,anti-CEA/anti-DPTA, anti-p185^(HER2)/anti-hapten; BsAbs as vaccineadjuvants; and BsAbs as diagnostic tools such as anti-rabbitIgG/anti-ferritin, anti-horse radish peroxidase (HRP)/anti-hormone,anti-somatostatin/anti-substance P, anti-HRP/anti-FITC,anti-CEA/anti-β-galactosidase. Examples of trispecific antibodiesinclude anti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 andanti-CD3/anti-CD8/anti-CD37. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

[0222] Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

[0223] According to a different approach, antibody variable domains withthe desired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

[0224] In a preferred embodiment of this approach, the bispecificantibodies are composed of a hybrid immunoglobulin heavy chain with afirst binding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in WO 94/04690. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986). According toanother approach described in W096/27011, the interface between a pairof antibody molecules can be engineered to maximize the percentage ofheterodimers which are recovered from recombinant cell culture. Thepreferred interface comprises at least a part of the C_(H)3 domain of anantibody constant domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

[0225] Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

[0226] Antibodies with more than two valencies are contemplated. Forexample, trispecific antibodies can be prepared. Tutt et al. J. Immunol.147: 60 (1991).

[0227] While the polypeptide of interest herein is preferably anantibody, other Fc region-containing polypeptides which can be modifiedaccording to the methods described herein are contemplated. An exampleof such a molecule is an immunoadhesin.

[0228] C. Immunoadhesin Preparation

[0229] The simplest and most straightforward immunoadhesin designcombines the binding domain(s) of the adhesin (e.g. the extracellulardomain (ECD) of a receptor) with the Fc region of an immunoglobulinheavy chain. Ordinarily, when preparing the immunoadhesins of thepresent invention, nucleic acid encoding the binding domain of theadhesin will be fused C-terminally to nucleic acid encoding theN-terminus of an immunoglobulin constant domain sequence, howeverN-terminal fusions are also possible.

[0230] Typically, in such fusions the encoded chimeric polypeptide willretain at least functionally active hinge, C_(H)2 and C_(H)3 domains ofthe constant region of an immunoglobulin heavy chain. Fusions are alsomade to the C-terminus of the Fc portion of a constant domain, orimmediately N-terminal to the C_(H)1 of the heavy chain or thecorresponding region of the light chain. The precise site at which thefusion is made is not critical; particular sites are well known and maybe selected in order to optimize the biological activity, secretion, orbinding characteristics of the immunoadhesin.

[0231] In a preferred embodiment, the adhesin sequence is fused to theN-terminus of the Fc region of immunoglobulin G₁(IgG₁). It is possibleto fuse the entire heavy chain constant region to the adhesin sequence.However, more preferably, a sequence beginning in the hinge region justupstream of the papain cleavage site which defines IgG Fc chemically(i.e. residue 216, taking the first residue of heavy chain constantregion to be 114), or analogous sites of other immunoglobulins is usedin the fusion. In a particularly preferred embodiment, the adhesin aminoacid sequence is fused to (a) the hinge region and C_(H)2 and C_(H)3 or(b) the C_(H)1, hinge, C_(H)2 and C_(H)3 domains, of an IgG heavy chain.

[0232] For bispecific immunoadhesins, the immunoadhesins are assembledas multimers, and particularly as heterodimers or heterotetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four chain unit is repeated in the highermolecular weight immunoglobulins; IgM generally exists as a pentamer offour basic units held together by disulfide bonds. IgA globulin, andoccasionally IgG globulin, may also exist in multimeric form in serum.In the case of multimer, each of the four units may be the same ordifferent.

[0233] Various exemplary assembled immunoadhesins within the scopeherein are schematically diagrammed below:

[0234] (a) AC_(L)-AC_(L);

[0235] (b) AC_(H)-(AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H));

[0236] (c) AC_(L)-AC_(H)-(AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),V_(L)C_(L)-AC_(H), or V_(L)C_(L)-V_(H)C_(H))

[0237] (d) AC_(L)-V_(H)C_(H)-(AC_(H), or AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H));

[0238] (e) V_(L)C_(L)-AC_(H)-(AC_(L)-V_(H)C_(H), or V_(L)C_(L)-AC_(H));and

[0239] (f) (A-Y)_(n)-(V_(L)C_(L)-V_(H)C_(H))₂,

[0240] wherein each A represents identical or different adhesin aminoacid sequences;

[0241] V_(L) is an immunoglobulin light chain variable domain;

[0242] V_(H) is an immunoglobulin heavy chain variable domain;

[0243] C_(L) is an immunoglobulin light chain constant domain;

[0244] C_(H) is an immunoglobulin heavy chain constant domain;

[0245] n is an integer greater than 1;

[0246] Y designates the residue of a covalent cross-linking agent.

[0247] In the interests of brevity, the foregoing structures only showkey features; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed tobe present in the ordinary locations which they occupy in theimmunoglobulin molecules.

[0248] Alternatively, the adhesin sequences can be inserted betweenimmunoglobulin heavy chain and light chain sequences, such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the adhesin sequences are fused to the 3′ end of animmunoglobulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the C_(H)2 domain, or between the C_(H)2 andC_(H)3 domains. Similar constructs have been reported by Hoogenboom, eta., Mol. Immunol. 28:1027-1037 (1991).

[0249] Although the presence of an immunoglobulin light chain is notrequired in the immunoadhesins of the present invention, animmunoglobulin light chain might be present either covalently associatedto an adhesin-immunoglobulin heavy chain fusion polypeptide, or directlyfused to the adhesin. In the former case, DNA encoding an immunoglobulinlight chain is typically coexpressed with the DNA encoding theadhesin-immunoglobulin heavy chain fusion protein. Upon secretion, thehybrid heavy chain and the light chain will be covalently associated toprovide an immunoglobulin-like structure comprising two disulfide-linkedimmunoglobulin heavy chain-light chain pairs. Methods suitable for thepreparation of such structures are, for example, disclosed in U.S. Pat.No. 4,816,567, issued 28 Mar. 1989.

[0250] Immunoadhesins are most conveniently constructed by fusing thecDNA sequence encoding the adhesin portion in-frame to an immunoglobulincDNA sequence. However, fusion to genomic immunoglobulin fragments canalso be used.(see, e.g. Aruffo et al., Cell 61:1303-1313 (1990); andStamenkovic et al., Cell 66:1133-1144 (1991)). The latter type of fusionrequires the presence of Ig regulatory sequences for expression. cDNAsencoding IgG heavy-chain constant regions can be isolated based onpublished sequences from cDNA libraries derived from spleen orperipheral blood lymphocytes, by hybridization or by polymerase chainreaction (PCR) techniques. The cDNAs encoding the “adhesin” and theimmunoglobulin parts of the immunoadhesin are inserted in tandem into aplasmid vector that directs efficient expression in the chosen hostcells.

[0251] D. Vectors, Host Cells and Recombinant Methods

[0252] The invention also provides isolated nucleic acid encoding apolypeptide variant as disclosed herein, vectors and host cellscomprising the nucleic acid, and recombinant techniques for theproduction of the polypeptide variant.

[0253] For recombinant production of the polypeptide variant, thenucleic acid encoding it is isolated and inserted into a replicablevector for further cloning (amplification of the DNA) or for expression.DNA encoding the polypeptide variant is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding thepolypeptide variant). Many vectors are available. The vector componentsgenerally include, but are not limited to, one or more of the following:a signal sequence, an origin of replication, one or more marker genes,an enhancer element, a promoter, and a transcription terminationsequence.

[0254] (i) Signal Sequence Component

[0255] The polypeptide variant of this invention may be producedrecombinantly not only directly, but also as a fusion polypeptide with aheterologous polypeptide, which is preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature protein or polypeptide. The heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process the native polypeptide variant signalsequence, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, Ipp, or heat-stable enterotoxin 11 leaders.For yeast secretion the native signal sequence may be substituted by,e.g., the yeast invertase leader, α factor leader (includingSaccharomyces and Kluyveromyces α-factor leaders), or acid phosphataseleader, the C. albicans glucoamylase leader, or the signal described inWO 90/13646. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

[0256] The DNA for such precursor region is ligated in reading frame toDNA encoding the polypeptide variant.

[0257] (ii) Origin of Replication Component

[0258] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Generally, in cloning vectors this sequence is one thatenables the vector to replicate independently of the host chromosomalDNA, and includes origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells. Generally, the origin of replication component is not needed formammalian expression vectors (the SV40 origin may typically be used onlybecause it contains the early promoter).

[0259] (iii) Selection Gene Component

[0260] Expression and cloning vectors may contain a selection gene, alsotermed a selectable marker. Typical selection genes encode proteins that(a) confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

[0261] One example of a selection scheme utilizes a drug to arrestgrowth of a host cell. Those cells that are successfully transformedwith a heterologous gene produce a protein conferring drug resistanceand thus survive the selection regimen. Examples of such dominantselection use the drugs neomycin, mycophenolic acid and hygromycin.

[0262] Another example of suitable selectable markers-for mammaliancells are those that enable the identification of cells competent totake up the polypeptide variant nucleic acid, such as DHFR, thymidinekinase, metallothionein-I and -II, preferably primate metallothioneingenes, adenosine deaminase, ornithine decarboxylase, etc.

[0263] For example, cells transformed with the DHFR selection gene arefirst identified by culturing all of the transformants in a culturemedium that contains methotrexate (Mtx), a competitive antagonist ofDHFR. An appropriate host cell when wild-type DHFR is employed is theChinese hamster ovary (CHO) cell line deficient in DHFR activity.

[0264] Alternatively, host cells (particularly wild-type hosts thatcontain endogenous DHFR) transformed or co-transformed with DNAsequences encoding polypeptide variant, wild-type DHFR protein, andanother selectable marker such as aminoglycoside 3′-phosphotransferase(APH) can be selected by cell growth in medium containing a selectionagent for the selectable marker such as an aminoglycosidic antibiotic,e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

[0265] A suitable selection gene for use in yeast is the trp1 genepresent in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39(1979)). The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of thetrtp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

[0266] In addition, vectors derived from the 1.6 μm circular plasmidpKD1 can be used for transformation of Kluyveromyces yeasts.Alternatively, an expression system for large-scale production ofrecombinant calf chymosin was reported for K lactis. Van den Berg,Bio/Technology, 8:135 (1990). Stable multi-copy expression vectors forsecretion of mature recombinant human serum albumin by industrialstrains of Kluyveromyces have also been disclosed. Fleer et al.,Bio/Technology, 9:968-975 (1991).

[0267] (iv) Promoter Component

[0268] Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to thepolypeptide variant nucleic acid. Promoters suitable for use withprokaryotic hosts include the phoA promoter, β-lactamase and lactosepromoter systems, alkaline phosphatase, a tryptophan (trp) promotersystem, and hybrid promoters such as the tac promoter. However, otherknown bacterial promoters are suitable. Promoters for use in bacterialsystems also will contain a Shine-Dalgarno (S.D.) sequence operablylinked to the DNA encoding the polypeptide variant.

[0269] Promoter sequences are known for eukaryotes. Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

[0270] Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phospho-fructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0271] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

[0272] Polypeptide variant transcription from vectors in mammalian hostcells is controlled, for example, by promoters obtained from the genomesof viruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

[0273] The early and late promoters of the SV40 virus are convenientlyobtained as an SV40 restriction fragment that also contains the SV40viral origin of replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

[0274] (v) Enhancer Element Component

[0275] Transcription of a DNA encoding the polypeptide variant of thisinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thepolypeptide variant-encoding sequence, but is preferably located at asite 5′ from the promoter.

[0276] (vi) Transcription Termination Component

[0277] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the polypeptide variant.One useful transcription termination component is the bovine growthhormone polyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

[0278] (vii) Selection and Transformation of Host Cells

[0279] Suitable host cells for cloning or expressing the DNA in thevectors herein are the prokaryote, yeast, or higher eukaryote cellsdescribed above. Suitable prokaryotes for this purpose includeeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacili such as B. subtilis and B. licheniformis (e.g., B.licheniformis 41 P disclosed in DD 266,710 published 12 Apr. 1989),Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E.coli cloning host is E. coli 294 (ATCC 31,446), although other strainssuch as E. coli B, E. coli X1776 (ATCC 31,537), and E. coliW3110 (ATCC27,325) are suitable. These examples are illustrative rather thanlimiting.

[0280] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forpolypeptide variant-encoding vectors. Saccharomyces cerevisiae, orcommon baker's yeast, is the most commonly used among lower eukaryotichost microorganisms. However, a number of other genera, species, andstrains are commonly available and useful herein, such asSchizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis,K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii(ATCC 24,178), K. waltii(ATCC 56,500), K. drosophilarum (ATCC36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226);Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234);Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis;and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[0281] Suitable host cells for the expression of glycosylatedpolypeptide variant are derived from multicellular organisms. Examplesof invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(frutfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

[0282] Plant cell cultures of cotton, corn, potato, soybean, petunia,tomato, and tobacco can also be utilized as hosts.

[0283] However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure. Examples of useful mammalian host cell lines aremonkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

[0284] Host cells are transformed with the above-described expression orcloning vectors for polypeptide variant production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

[0285] (viii) Culturing the Host Cells

[0286] The host cells used to produce the polypeptide variant of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium((DMEM), Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham et al., Meth. Enz. 58:44 (1979),Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704;4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195;or U.S. Pat. No. Re. 30,985 may be used as culture media for the hostcells. Any of these media may be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0287] (ix) Polypeptide Variant Purification

[0288] When using recombinant techniques, the polypeptide variant can beproduced intracellularly, in the periplasmic space, or directly secretedinto the medium. If the polypeptide variant is produced intracellularly,as a first step, the particulate debris, either host cells or lysedfragments, is removed, for example, by centrifugation orultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992)describe a procedure for isolating antibodies which are secreted to theperiplasmic space of E. coli. Briefly, cell paste is thawed in thepresence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris canbe removed by centrifugation. Where the polypeptide variant is secretedinto the medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

[0289] The polypeptide variant composition prepared from the cells canbe purified using, for example, hydroxylapatite chromatography, gelelectrophoresis, dialysis, and affinity chromatography, with affinitychromatography being the preferred purification technique. Thesuitability of protein A as an affinity ligand depends on the speciesand isotype of any immunoglobulin Fc region that is present in thepolypeptide variant. Protein A can be used to purify polypeptidevariants that are based on human γ1, γ2, or γ4 heavy chains (Lindmark etal., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for allmouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575(1986)). The matrix to which the affinity ligand is attached is mostoften agarose, but other matrices are available. Mechanically stablematrices such as controlled pore glass or poly(styrenedivinyl)benzeneallow for faster flow rates and shorter processing times than can beachieved with agarose. Where the polypeptide variant comprises a C_(H)3domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) isuseful for purification. Other techniques for protein purification suchas fractionation on an ion-exchange column, ethanol precipitation,Reverse Phase HPLC, chromatography on silica, chromatography on heparinSEPHAROSE™ chromatography on an anion or cation exchange resin (such asa polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the polypeptidevariant to be recovered.

[0290] Following any preliminary purification step(s), the mixturecomprising the polypeptide variant of interest and contaminants may besubjected to low pH hydrophobic interaction chromatography using anelution buffer at a pH between about 2.5-4.5, preferably performed atlow salt concentrations (e.g.,from about 0-0.25M salt).

[0291] E. Pharmaceutical Formulations

[0292] Therapeutic formulations of the polypeptide variant are preparedfor storage by mixing the polypeptide variant having the desired degreeof purity with optional physiologically acceptable carriers, excipientsor stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0293] The formulation herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. Such molecules are suitably present in combination inamounts that are effective for the purpose intended.

[0294] The active ingredients may also be entrapped in microcapsuleprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0295] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes.

[0296] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the polypeptide variant, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

[0297] F. Non-Therapeutic Uses for the Polypeptide Variant

[0298] The polypeptide variant of the invention may be used as anaffinity purification agent. In this process, the polypeptide variant isimmobilized on a solid phase such a Sephadex resin or filter paper,using methods well known in the art. The immobilized polypeptide variantis contacted with a sample containing the antigen to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except the antigento be purified, which is bound to the immobilized polypeptide variant.Finally, the support is washed with another suitable solvent, such asglycine buffer, pH 5.0, that will release the antigen from thepolypeptide variant.

[0299] The polypeptide variant may also be useful in diagnostic assays,e.g., for detecting expression of an antigen of interest in specificcells, tissues, or serum.

[0300] For diagnostic applications, the polypeptide variant typicallywill be labeled with a detectable moiety. Numerous labels are availablewhich can be generally grouped into the following categories:

[0301] (a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. Thepolypeptide variant can be labeled with the radioisotope using thetechniques described in Current Protocols in Immunology, Volumes 1 and2, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991)for example and radioactivity can be measured using scintillationcounting.

[0302] (b) Fluorescent labels such as rare earth chelates (europiumchelates) or fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, Lissamine, phycoerythrin and Texas Red areavailable. The fluorescent labels can be conjugated to the polypeptidevariant using the techniques disclosed in Current Protocols inImmunology, supra, for example. Fluorescence can be quantified using afluorimeter.

[0303] (c) Various enzyme-substrate labels are available and U.S. Pat.No. 4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate that can bemeasured using various techniques. For example, the enzyme may catalyzea color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York, 73:147-166 (1981).

[0304] Examples of enzyme-substrate combinations include, for example:

[0305] (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′, 5,5′-tetramethyl benzidinehydrochloride (TMB));

[0306] (ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and

[0307] (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate(e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

[0308] Numerous other enzyme-substrate combinations are available tothose skilled in the art. For a general review of these, see U.S. Pat.Nos. 4,275,149 and 4,318,980.

[0309] Sometimes, the label is indirectly conjugated with thepolypeptide variant. The skilled artisan will be aware of varioustechniques for achieving this. For example, the polypeptide variant canbe conjugated with biotin and any of the three broad categories oflabels mentioned above can be conjugated with avidin, or vice versa.Biotin binds selectively to avidin and thus, the label can be conjugatedwith the polypeptide variant in this indirect manner. Alternatively, toachieve indirect conjugation of the label with the polypeptide variant,the polypeptide variant is conjugated with a small hapten (e.g.,digoxin) and one of the different types of labels mentioned above isconjugated with an anti-hapten polypeptide variant (e.g., anti-digoxinantibody). Thus, indirect conjugation of the label with the polypeptidevariant can be achieved.

[0310] In another embodiment of the invention, the polypeptide variantneed not be labeled, and the presence thereof can be detected using alabeled antibody which binds to the polypeptide variant.

[0311] The polypeptide variant of the present invention may be employedin any known assay method, such as competitive binding assays, directand indirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press,Inc. 1987).

[0312] The polypeptide variant may also be used for in vivo diagnosticassays. Generally, the polypeptide variant is labeled with aradionuclide (such as ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) sothat the antigen or cells expressing it can be localized usingimmunoscintiography.

[0313] G. In Vivo Uses for the Polypeptide Variant

[0314] It is contemplated that the polypeptide variant of the presentinvention may be used to treat a mammal e.g. a patient suffering from,or predisposed to, a disease or disorder who could benefit fromadministration of the polypeptide variant. The conditions which can betreated with the polypeptide variant are many and include cancer (e.g.where the polypeptide variant binds the HER2 receptor, CD20 or vascularendothelial growth factor (VEGF)); allergic conditions such as asthma(with an anti-IgE antibody); and LFA-1 -mediated disorders (e.g. wherethe polypeptide variant is an anti-LFA-1 or anti-ICAM-1 antibody) etc.

[0315] Where the antibody binds the HER2 receptor, the disorderpreferably is HER2-expressing cancer, e.g. a benign or malignant tumorcharacterized by overexpression of the HER2 receptor. Such cancersinclude, but are not limited to, breast cancer, squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, bladder cancer, hepatoma, colon cancer, colorectal cancer,endometrial carcinoma, salivary gland carcinoma, kidney cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer. According to theteachings herein, one may prepare a polypeptide with a variant Fc regionwhich has improved, or diminished, ADCC activity. Such molecules willfind applications in the treatment of different disorders.

[0316] For example, the polypeptide variant with improved ADCC activitymay be employed in the treatment of diseases or disorders wheredestruction or elimination of tissue or foreign micro-organisms isdesired. For example, the polypeptide may be used to treat cancer;inflammatory disorders; infections (e.g. bacterial, viral, fungal oryeast infections); and other conditions (such as goiter) where removalof tissue is desired, etc.

[0317] Where the polypeptide variant has diminished ADCC activity, suchvariants may be used to treat diseases or disorders where a Fcregion-containing polypeptide with long half-life is desired, but thepolypeptide preferably does not have undesirable effector function(s).For example, the Fc region-containing polypeptide may be an anti-tissuefactor (TF) antibody; anti-IgE antibody; and anti-integrin antibody(e.g. an anti-α4β7 antibody). The desired mechanism of action of such Fcregion-containing polypeptides may be to block ligand-receptor bindingpairs. Moreover, the Fc-region containing polypeptide with diminishedADCC activity may be an agonist antibody.

[0318] The polypeptide variant is administered by any suitable means,including parenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local immunosuppressive treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the polypeptide variant issuitably administered by pulse infusion, particularly with decliningdoses of the polypeptide variant. Preferably the dosing is given byinjections, most preferably intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.

[0319] For the prevention or treatment of disease, the appropriatedosage of polypeptide variant will depend on the type of disease to betreated, the severity and course of the disease, whether the polypeptidevariant is administered for preventive or therapeutic purposes, previoustherapy, the patient's clinical history and response to the polypeptidevariant, and the discretion of the attending physician. The polypeptidevariant is suitably administered to the patient at one time or over aseries of treatments.

[0320] Depending on the type and severity of the disease, about 1 μg/kgto 15 mg/kg (e.g., 0.1-20mg/kg) of polypeptide variant is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

[0321] The polypeptide variant composition will be formulated, dosed,and administered in a fashion consistent with good medical practice.Factors for consideration in this context include the particulardisorder being treated, the particular mammal being treated, theclinical condition of the individual patient, the cause of the disorder,the site of delivery of the agent, the method of administration, thescheduling of administration, and other factors known to medicalpractitioners. The “therapeutically effective amount” of the polypeptidevariant to be administered will be governed by such considerations, andis the minimum amount necessary to prevent, ameliorate, or treat adisease or disorder. The polypeptide variant need not be, but isoptionally formulated with one or more agents currently used to preventor treat the disorder in question. The effective amount of such otheragents depends on the amount of polypeptide variant present in theformulation, the type of disorder or treatment, and other factorsdiscussed above. These are generally used in the same dosages and withadministration routes as used hereinbefore or about from 1 to 99% of theheretofore employed dosages.

[0322] The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of this invention. All literature and patent citationsmentioned herein are expressly incorporated by reference.

EXAMPLE 1 Low Affinity Receptor Binding Assay

[0323] This assay determines binding of an IgG Fc region to recombinantFcγRIIA, FcγRIIB and FcγRIIIA α subunits expressed as His6-glutathione Stransferase (GST)-tagged fusion proteins. Since the affinity of the Fcregion of IgG1for the FcγRI is in the nanomolar range, the binding ofIgG1Fc variants can be measured by titrating monomeric IgG and measuringbound IgG with a polyclonal anti-IgG in a standard ELISA format (Example2 below). The affinity of the other members of the FcγR family, i.e.FcγRIIA, FcγRIIB and FcγRIIIA for IgG is however in the micromolar rangeand binding of monomeric IgG1for these receptors can not be reliablymeasured in an ELISA format.

[0324] The following assay utilizes Fc variants of recombinant anti-IgEE27 (FIGS. 4A and 4B) which, when mixed with human IgE at a 1:1 molarratio, forms a stable hexamer consisting of three anti-IgE molecules andthree IgE molecules. A recombinant chimeric form of IgE (chimeric IgE)was engineered and consists of a human IgE Fc region and the Fab of ananti-VEGF antibody (Presta et al. Cancer Research 57:4593-4599 (1997))which binds two VEGF molecules per mole of anti-VEGF. When recombinanthuman VEGF is added at a 2:1 molar ratio to chimeric IgE:E27 hexamers,the hexamers are linked into larger molecular weight complexes via thechimeric IgE Fab:VEGF interaction. The E27 component of this complexbinds to the FcγRIIA, FcγRIIB and FcγRIIIA α subunits with higheravidity to permit detection in an ELISA format.

Materials and Methods

[0325] Receptor Coat Fc γ receptor α subunits were expressed as GSTfusions of His6 tagged extracellular domains (ECDs) in 293 cellsresulting in an ECD-6His-GST fusion protein (Graham et al. J. Gen.Virol. 36:59-74 (1977) and Gorman et al. DNA Prot. Eng. Tech. 2:3-10(1990)) and purified by Ni-NTA column chromatography (Qiagen, Australia)and buffer exchanged into phosphate buffered saline (PBS).Concentrations were determined by absorption at 280 nm using extinctioncoefficients derived by amino acid composition analysis. Receptors werecoated onto Nunc F96 maxisorb plates (cat no. 439454) at 100 ng per wellby adding 100 μl of receptor-GST fusion at 1 μg/ml in PBS and incubatedfor 48 hours at 4° C. Prior to assay, plates are washed 3× with 250 μlof wash buffer (PBS pH 7.4 containing 0.5% TWEEN 20™) and blocked with250 μl of assay buffer (50 mM Tris buffered saline, 0.05% TWEEN 20™,0.5% RIA grade bovine albumin (Sigma A7888), and 2mM EDTA pH 7.4).

[0326] Immune Complex Formation: Equal molar amounts (1:1) of E27 andrecombinant chimeric IgE which binds two moles recombinant human VEGFper mole of chimeric IgE are added to a 12×75 mm polypropylene tube inPBS and mixed by rotation for 30 minutes at 25° C. E27(anti-IgE)/chimeric IgE (IgE) hexamers are formed during thisincubation. Recombinant human VEGF (165 form, MW 44,000) is added at a2:1 molar ratio to the IgE concentration and mixed by rotation anadditional 30 minutes at 25° C. VEGF-chimeric IgE binding linksE27:chimeric IgE hexamers into larger molecular weight complexes whichbind FcγR α subunit ECD coated plates via the Fc region of the E27antibody.

[0327] E27:chimeric IgE:VEGF: (1:1:2 molar ratio) complexes are added toFcγR α subunit coated plates at E27 concentrations of 5 μg and 1 μgtotal IgG in quadruplicate in assay buffer and incubated for 120 minutesat 25° C. on an orbital shaker.

[0328] Complex Detection: Plates are washed 5×with wash buffer to removeunbound complexes and IgG binding is detected by adding 100 μl horseradish peroxidase (HRP) conjugated goat anti-human IgG (γ) heavy chainspecific (Boehringer Mannheim 1814249) at 1:10,000 in assay buffer andincubated for 90 min at 25° C. on an orbital shaker. Plates are washed5×with wash buffer to remove unbound HRP goat anti-human IgG and boundanti-IgG is detected by adding 100 μl of substrate solution (0.4mg/mlo-phenylenedaimine dihydrochloride, Sigma P6912, 6 mM H₂O₂ in PBS) andincubating for 8 min at 25° C. Enzymatic reaction is stopped by theaddition of 100 μl 4.5N H₂SO₄ and colorimetric product is measured at490 nm on a 96 well plate densitometer (Molecular Devices). Binding ofE27 variant complexes is expressed as a percent of the wild type E27containing complex.

EXAMPLE 2 Identification of Unique C1q Binding Sites in a Human IgGAntibody

[0329] In the present study, mutations were identified in the CH2 domainof a human IgG1antibody, “C2B8” (Reff et al., Blood 83:435 (1994)), thatablated binding of the antibody to C1q but did not alter theconformation of the antibody nor affect binding to each of the FcγRs. Byalanine scanning mutagenesis, five variants in human IgG1 wereidentified, D270K, D270V, K322A P329A, and P331, that were non-lytic andhad decreased binding to C1q. The data suggested that the core C1qbinding sites in human IgG1 is different from that of murine IgG2b. Inaddition, K322A, P329A and P331A were found to bind normally to the CD20antigen, and to four Fc receptors, FcγRI, FcγRII, FcγRIII and FcRn.

Materials and Methods

[0330] Construction of C2B8 Variants: The chimeric light and heavychains of anti-CD20 antibody C2B8 (Reff et al., Blood 83:435 (1994))subcloned separately into previously described PRK vectors (Gorman etal., DNA Protein Eng. Tech. 2:3 (1990)) were used. By site directedmutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488 (1985)),alanine scan variants of the Fc region in the heavy chain wereconstructed. The heavy and light chain plasmids were co-transfected intoan adenovirus transformed human embryonic kidney cell line as previouslydescribed (Werther et al., J. Immunol. 157:4986 (1996)). The media waschanged to serum-free 24 hours after transfection and the secretedantibody was harvested after five days. The antibodies were purifiedusing Protein A-SEPHAROSE CL-4B™ (Pharmacia), buffer exchanged andconcentrated to 0.5 ml with PBS using a Centricon-30 (Amicon), andstored at 4° C. The concentration of the antibody was determined usingtotal Ig-binding ELISA.

[0331] C1q Binding ELISA: Costar 96 well plates were coated overnight at4° C. with the indicated concentrations of C2B8 in coating buffer (0.05M sodium carbonate buffer), pH 9. The plates were then washed 3× withPBS/0.05% TWEEN 20™, pH 7.4 and blocked with 200 μl of ELISA diluentwithout thimerosal (0.1M NaPO4 /0.1M NaCl/0.1% gelatin /0.05% TWEEN20™/0.05% ProClin300) for 1 hr at room temperature. The plate was washed3× with wash buffer, an aliquot of 100 μl of 2 μg/ml C1q (Quidel, SanDiego, Calif.) was added to each well and incubated for 2 hrs at roomtemperature. The plate was then washed 6×with wash buffer. 100 μl of a1:1000 dilution of sheep anti-complement C1q peroxidase conjugatedantibody (Biodesign) was added to each well and incubated for 1 hour atroom temperature. The plate was again washed 6×with wash buffer and 100μl of substrate buffer (PBS/0.012% H₂0₂) containing OPD(O-phenylenediamine dihydrochloride (Sigma)) was added to each well. Theoxidation reaction, observed by the appearance of a yellow color, wasallowed to proceed for 30 minutes and stopped by the addition of 100 μlof 4.5 N H₂SO₄. The absorbance was then read at (492-405) nm using amicroplate reader (SPECTRA MAX 250™, Molecular Devices Corp.). Theappropriate controls were run in parallel (i.e. the ELISA was performedwithout C1q for each concentration of C2B8 used and also the ELISA wasperformed without C2B8). For each variant, C1q binding was measured byplotting the absorbance (492-405) nm versus concentration of C2B8 inμg/ml using a 4-parameter curve fitting program (KALEIDAGRAPH™) andcomparing EC₅₀ values.

[0332] Complement Dependent Cytotoxicity (CDC) Assay: This assay wasperformed essentially as previously described (Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1997)). Various concentrations of C2B8(0.08-20 μg/ml) were diluted with RHB buffer (RPMI 1640/20 mM HEPES (pH7.2)/2 mM Glutamine/0.1% BSA/100 μg/ml Gentamicin). Human complement(Quidel) was diluted 1:3 in RHB buffer and WIL2-S cells (available fromthe ATCC, Manassas, Va.) which express the CD20 antigen were diluted toa density of 1×10⁶ cells/ml with RHB buffer. Mixtures of 150 μlcontaining equal volumes of C2B8, diluted human complement and WIL2-Scells were added to a flat bottom tissue culture 96 well plate andallowed to incubate for 2 hrs at 37° C. and 5% CO₂ to facilitatecomplement mediated cell lysis. 50 μl of alamar blue (AccumedInternational) was then added to each well and incubated overnight at37° C. The absorbance was measured using a 96-well fluorometer withexcitation at 530 nm and emission at 590 nm. As described byGazzano-Santoro et al., the results are expressed in relativefluorescence units (RFU). The sample concentrations were computed from aC2B8 standard curve and the percent activity as compared to wild typeC2B8 is reported for each variant.

[0333] CD20 Binding Potency of the C2B8 Variants: The binding of C2B8and variants to the CD20 antigen were assessed by a method previouslydescribed (Reff et al., (1994), supra; reviewed in Gazzano-Santoro etal., (1997), supra). WIL2-S cells were grown for 3-4 days to a celldensity of 1×10⁶ cells/ml. The cells were washed and spun twice in FACSbuffer (PBS/0.1% BSA/0.02% NaN₃) and resuspended to a cell density of5×10⁶ cells/ml. 200 μl of cells (5×10⁶ cells/ml) and 20 μl of dilutedC2B8 samples were added to a 5 ml tube and incubated at room temperaturefor 30 minutes with agitation. The mixture was then washed with 2 ml ofcold FACS buffer, spun down and resuspended in 200 μl of cold FACSbuffer. To the suspension, 10 μl of goat anti-human IgG-FITC (AmericanQualex Labs.) was added and the mixture was incubated in the dark atroom temperature for 30 minutes with agitation. After incubation, themixture was washed with 2 ml of FACS buffer, spun down and resuspendedin 1 ml of cold fixative buffer (1% formaldehyde in PBS). The sampleswere analyzed by flow cytometry and the results expressed as relativefluorescence units (RFU) were plotted against antibody concentrationsusing a 4-parameter curve fitting program (KALEIDAGRAPH™). The EC₅₀values are reported as a percentage of that of the C2B8 referencematerial.

[0334] FcγR Binding ELISAs: FcγRI α subunit-GST fusion was coated ontoNunc F96 maxisorb plates (cat no. 439454) by adding 100 μl ofreceptor-GST fusion at 1 μg/ml in PBS and incubated for 48 hours at 4°C. Prior to assay, plates are washed 3× with 250 μl of wash buffer (PBSpH 7.4 containing 0.5% TWEEN 20™) and blocked with 250 μl of assaybuffer (50 mM Tris buffered saline, 0.05% TWEEN 20™, 0.5% RIA gradebovine albumin (Sigma A7888), and 2 mM EDTA pH 7.4). Samples diluted to10 μg/ml in 1 ml of assay buffer are added to FcγRI α subunit coatedplates and incubated for 120 minutes at 25° C. on an orbital shaker.Plates are washed 5×with wash buffer to remove unbound complexes and IgGbinding is detected by adding 100 μl horse radish peroxidase (HRP)conjugated goat anti-human IgG (γ) heavy chain specific (BoehringerMannheim 1814249) at 1:10,000 in assay buffer and incubated for 90 minat 25° C. on an orbital shaker. Plates are washed 5×with wash buffer toremove unbound HRP goat anti-human IgG and bound anti-IgG is detected byadding 100 μl of substrate solution (0.4 mg/ml o-phenylenedaiminedihydrochloride, Sigma P6912, 6 mM H₂O₂ in PBS) and incubating for 8 minat 25° C. Enzymatic reaction is stopped by the addition of 100 μl 4.5NH₂SO₄ and calorimetric product is measured at 490 nm on a 96 well platedensitometer (Molecular Devices). Binding of variant is expressed as apercent of the wild type molecule.

[0335] FcγRII and III binding ELISAs were performed as described inExample 1 above.

[0336] For measuring FcRn binding activity of IgG variants, ELISA plateswere coated with 2 μg/ml streptavidin (Zymed, South San Francisco) in 50mM carbonate buffer, pH 9.6, at 4° C. overnight and blocked withPBS-0.5% BSA, pH 7.2 at room temperature for one hour. Biotinylated FcRn(prepared using biotin-X-NHS from Research Organics, Cleveland, Ohio andused at 1-2 μg/ml) in PBS-0.5% BSA, 0.05% polysorbate 20, pH 7.2, wasadded to the plate and incubated for one hour. Two fold serial dilutionsof IgG standard (1.6-100 ng/ml) or variants in PBS-0.5% BSA, 0.05%polysorbate 20, pH 6.0, were added to the plate and incubated for twohours. Bound IgG was detected using peroxidase labeled goat F(ab′)₂anti-human IgG F(ab′)₂ in the above pH 6.0 buffer (JacksonImmunoResearch, West Grove, Pa.) followed by 3,3′,5,5′-tetramethylbenzidine (Kirgaard & Perry Laboratories) as the substrate. Plates werewashed between steps with PBS-0.05% polysorbate 20 at either pH 7.2 or6.0. Absorbance was read at 450 nm on a Vmax plate reader (MolecularDevices, Menlo Park, Calif.). Titration curves were fit with afour-parameter nonlinear regression curve-fitting program (KaleidaGraph,Synergy software, Reading, Pa.). Concentrations of IgG variantscorresponding to the mid-point absorbance of the titration curve of thestandard were calculated and then divided by the concentration of thestandard corresponding to the mid-point absorbance of the standardtitration curve.

Results and Discussion

[0337] By alanine scanning mutagenesis, several single point mutationswere constructed in the CH2 domain of C2B8 beginning with E318A, K320Aand K322A. All the variants constructed bound normally to the CD20antigen (Table 3). TABLE 3 wt E318A K320A K322A P329A P331A FcRn + + + +CD20 + + + + + + FcγRI + + + + + + FcγRII + + + + + +FcγRIII + + + + + + *C1q +++ ++ +++ − − − CDC + + + − − −

[0338] Where binding of human complement to an antibody with a human Fcwas analyzed, the ability of E318A and K320A to activate complement wasessentially identical to that of wild type C2B8 (Table 3). When comparedto wild type C2B8, there appears to be little difference in the bindingof E318A and K320A to C1q. There is only a 10% decrease in the bindingof K320A and about a 30% decrease in the binding of E318A to C1q (FIG.2). The results indicate that the effect of the E318A and the K320Asubstitution on complement activation and C1q binding is minimal. Also,the human IgG1of C2B8 was substituted for human IgG2 and used as anegative control in'the C1q binding studies. The IgG2 variant appears tohave a much lower affinity for C1q than the E318A and K320A variants(FIG. 2). Thus, the results demonstrate that E318 and K320 do notconstitute the core C1q binding sites for human IgG1. Conversely, theK322A substitution had a significant effect on both complement activityand C1q binding. The K322A variant did not have CDC activity when testedin the above CDC assay and was more than a 100 fold lower than wild typeC2B8 in binding to C1q (FIG. 2). In the human system, K322 is the onlyresidue of the proposed core C1q binding sites that appeared to have asignificant effect on complement activation and C1q binding.

[0339] Since the Duncan and Winter study was performed using mouse IgG2band the above results reveal that K320 and E318 in human IgG1 are notinvolved in C1q binding, and without being bound to any one theory, theabove data suggest that the C1q binding region in murine IgGs isdifferent from that of the human. To investigate this further and alsoto identify additional variants that do not bind to C1q and hence do notactivate complement, several more point mutations in the vicinity ofK322 were constructed as assessed from the three dimensional structureof the C2B8 Fc. Variants constructed, K274A, N276A, Y278A, S324A, P329A,P331A, K334A, and T335A, were assessed for their ability to bind C1q andalso to activate complement. Many of these substitutions had little orno effect on C1q binding or complement activation. In the above assays,the P329A and the P331A variants did not activate complement and haddecreased binding to C1q. The P331A variant did not activate complementand was 60 fold lower in binding to C1q (FIG. 3) when compared to wildtype C2B8 (FIG. 2). The concentration range of the antibody variantsused in FIG. 3 is expanded to 100 μg/ml in order to observe saturationof C1q binding to the P331A variant. The mutation P329A results in anantibody that does not activate complement and is more than a 100 foldlower in binding to C1q (FIG. 3) when compared to wild type C2B8 (FIG.2).

[0340] Variants that did not bind to C1q and hence did not activatecomplement were examined for their ability to bind to the Fc receptors:FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA and FcRn. This particular study wasperformed using a humanized anti-IgE antibody, an IgG1antibody withthese mutations (see Example 1 above). The results revealed thevariants, K322A and P329A, bind to all the Fc receptors to the sameextent as the wild type protein (Table 4). However, there was a slightdecrease in the binding of P331A to FcγRIIB.

[0341] In conclusion, two amino acid substitutions in the COOH terminalregion of the CH2 domain of human IgG₁, K322A and P329A were identifiedthat result in more than 100 fold decrease in C1q binding and do notactivate the CDC pathway. These two variants, K322A and P329A, bind toall Fc receptors with the same affinity as the wild type antibody. Basedon the results, summarized in Table 4, and without being bound to anyone theory, it is proposed that the C1q binding epicenter of humanIgG1is centered around K322, P329 and P331 and is different from themurine IgG2b epicenter which constitutes E318, K320 and K322. TABLE 4 wtE318A K320A K322A P329A P331A CD20 100 89 102 86 112 103 ^(a)FcγRI 10093 102 90 104 74 ^(a)FcγRIIA 100 113 94 109 111 86 ^(a)FcγRIIB 100 10683 101 96 58 ^(a)FcγRIII 100 104 72 90 85 73 CDC 100 108 108 none nonenone

[0342] A further residue involved in binding human C1q was identifiedusing the methods described in the present example. The residue D270 wasreplaced with lysine and valine to generate variants D270K and D270V,respectively. These variants both showed decreased binding to human C1q(FIG. 6) and were non-lytic (FIG. 7). The two variants bound the CD20antigen normally and recruited ADCC.

EXAMPLE 3 Variants with Improved C1q Binding

[0343] The following study shows that substitution of residues atpositions K326, A327, E333 and K334 resulted in variants with at leastabout a 30% increase in binding to C1q when compared to the wild typeantibody. This indicated K326, A327, E333 and K334 are potential sitesfor improving the efficacy of antibodies by way of the CDC pathway. Theaim of this study was to improve CDC activity of an antibody byincreasing binding to C1q. By site directed mutagenesis at K326 andE333, several variants with increased binding to C1q were constructed.The residues in order of increased binding at K326 are K<V<E<A<G<D<M<W,and the residues in order of increased binding at E333 areE<Q<D<V<G<A<S. Four variants, K326M, K326D, K326E and E333S wereconstructed with at least a two-fold increase in binding to C1q whencompared to wild type. Variant K326W displayed about a five-foldincrease in binding to C1q.

[0344] Variants of the wild type C2B8 antibody were prepared asdescribed above in Example 2. A further control antibody, wild type C2B8produced in Chinese hamster ovary (CHO) cells essentially as describedin U.S. Pat. No. 5,736,137, was included in a C1q binding ELISA toconfirm that wt C2B8 produced in the 293 kidney cell line had the sameC1q binding activity as the CHO-produced antibody (see “CHO-wt-C2B8” inFIG. 8). The C1q binding ELISA, CDC assay, and CD20 binding potencyassay in this example were performed as described in Example 2 above.

[0345] As shown in FIG. 8, alanine substitution at K326 and E333 in C2B8resulted in variants with about a 30% increase in binding to C1q.

[0346] Several other single point variants at K326 and E333 wereconstructed and assessed for their ability to bind C1q and activatecomplement. All the variants constructed bound normally to the CD20antigen.

[0347] With respect to K326, the other single point variants constructedwere K326A, K326D, K326E, K326G, K326V, K326M and K326W. As shown inFIG. 9, these variants all bound to C1q with a better affinity than thewild type antibody. K326W, K326M, K326D and K326E showed at least atwo-fold increase in binding to C1q (Table 5). Among the K326 variants,K326W had the best affinity for C1q. TABLE 5 Variant EC₅₀ value Wildtype 1.53 K326V 1.30 K326A 1.03 K326E 1.08 K326G 0.95 K326D 0.76 K326M0.67 K326W 0.47 E333S 0.81 E333A 0.98 E333G 1.14 E333V 1.18 E333D 1.22E333Q 1.52 K334A 1.07

[0348] Substitutions with hydrophobic as well as charged residuesresulted in variants with increased binding to C1q. Even substitutionwith glycine which is known to impart flexibility to a chain and is wellconserved in nature, resulted in a variant with higher affinity for C1qwhen compared to the wild type. It would appear that any amino acidsubstitution at this site would result in a variant with higher affinityfor C1q. As assessed from the three-dimensional structure, K326 and E333are in the vicinity of the core C1q binding sites (FIG. 10).

[0349] In addition to alanine, E333 was also substituted with otheramino acid residues. These variants, E333S, E333G, E333V, E333D, andE333Q, all had increased binding to C1q when compared to the wild type(FIG. 11). As shown in Table 5, the order of binding affinity for C1qwas as follows: E333S>E333A>E333G>E333V>E333D>E333Q. Substitutions withamino acid residues with small side chain volumes, i.e. serine, alanineand glycine, resulted in variants with higher affinity for C1q incomparison to the other variants, E333V, E333D and E333Q, with largerside chain volumes. The variant E333S had the highest affinity for C1q,showing a two-fold increase in binding when compared to the wild type.Without being bound to anyone theory, this indicates the effect on C1qbinding at 333 may also be due in part to the polarity of the residue.

[0350] Double variants were also generated. As shown in FIGS. 12 and 13,double variants K326M-E333S and K326A-E333A were at least three-foldbetter at binding human C1q than wild type C2B8 (FIG. 12) and at leasttwo-fold better at mediating CDC compared to wild type C2B8 (FIG. 13).Additivity indicates these are independently acting variants.

[0351] As shown in FIG. 14, a further variant with improved C1q binding(50% increase) was made by changing A327 in a human IgG1 constant regionto glycine. Conversely, in a human IgG2 constant region, changing G327to alanine reduced C1q binding of the IgG2 antibody.

EXAMPLE 4 Identification of FcR Binding Sites in Human IgG Antibodies

[0352] In the present study, the effect of mutating various Fc regionresidues of an IgG1antibody with respect to binding FcγRI, FcγRIIA,FcγRIIB and FcγRIIIIA as well as FcRn was evaluated. Antibody variantswith improved as well as diminished FcR binding were identified.

Materials and Methods

[0353] Construction of IgG1Variants: Recombinant anti-IgE E27 having thelight chain and heavy chain sequences in FIGS. 4A and 4B, respectively,was used as the parent antibody in the following experiments. Thisantibody binds the antigen IgE and has a non-A allotype IgG1Fc region.By site directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA82:488 (1985)), variants of the Fc region in the heavy chain of theabove parent antibody were constructed. The heavy and light chainplasmids were co-transfected into an adenovirus transformed humanembryonic kidney cell line as previously described (Werther et al., J.Immunol. 157:4986 (1996)). The media was changed to serum-free 24 hoursafter transfection and the secreted antibody was harvested after fivedays. The antibodies were purified by Protein G SEPHAROSE® (Pharmacia),buffer exchanged and concentrated to 0.5 ml with PBS using aCentricon-30 (Amicon), and stored at 4° C. Concentration was determinedby adsorption at 280 nm using extinction coefficients derived by aminoacid composition analysis.

[0354] High Affinity FcγRIA Binding ELISA: FcγRIA was expressed as a GSTfusion of His6 tagged extracellular domain in 293 cells and purified byNi-NTA column chromatography.

[0355] To purify FcγRIA, supernatant from transfected 293 cells wasremoved after three days. Protease inhibitors were added; 50 μLAprotinin (Sigma)/50 mL supernatant, and PMSF (1 mM). Supernatants wereconcentrated to 10 mL in a stirred cell (Amicon), and dialyzed overnightat 4° C. against 1 liter column buffer (50 mM Tris pH 8.0, 20 mMImidazole, 300 mM NaCl). Additional dialysis was done the followingmorning against fresh column buffer for 4 hours at 4° C. The solutionwas loaded on to a 1 mL Ni⁺⁺ column (NTA super flow resin, Qiagen)previously equilibrated with 10 mL column buffer. Columns were washedwith 10 mL column buffer, and protein was eluted with 2.5 mL elutionbuffer (50 mM Tris pH 8.0, 250 mM Imidazole, 300 mM NaCl). Protein wasconcentrated to 0.5 mL and buffer exchanged into PBS. Concentrationswere determined by adsorption at 280 nm using an extinction coefficientderived by amino acid composition analysis.

[0356] Purified receptors were coated onto Nunc F96 maxisorb plates (catno. 439545) at approximately 150 ng per well by adding 100 μL ofreceptor at 1.5 μg/mL in PBS and incubated for 24 hours at 4° C. Priorto assay, plates were washed 3× with 250 μL of wash buffer (phosphatebuffered saline pH 7.4 containing 0.5% TWEEN 20®) and blocked with 250μL of assay buffer (50 mM tris buffered saline, 0.05% TWEEN 20®, 0.5%RIA grade bovine albumin (Sigma A7888), and 2 mM EDTA pH 7.4).

[0357] 100 μL of E27 was added to the first four wells of the FcγRIAsubunit coated plated at a concentration of 10 μg/mL. 80 μL of assaybuffer was added to the next four well followed by 20 μL of the 10 μg/mLE27 IgG to give a final concentration of 2 μg/mL. Plates were incubatedat 25° C. for 2 hours on an orbital shaker.

[0358] For detection, plates were washed 5×with wash buffer to removeunbound antibody. IgG binding to GST-FcγRIA was detected by adding 100μL horse radish peroxidase (HRP) conjugated protein G (BIORAD) at1:5000. HRP conjugates were incubated for 1.5 hours at 25° C. on anorbital shaker. Plates were washed ×5 with wash buffer to remove unboundHRP conjugate. Binding was detected by adding 100 μL of substratesolution (0.4 mg/mL o-phenylenedaimine dihydrochloride, Sigma P6912, 6mM H₂O₂ in PBS) and incubating for 10 minutes at 25° C. Enzymaticreaction was stopped by the addition of 100 μL of 4.5 N H₂SO₄ andcolorimetric product was measured at 490 nm on a 96 well platedensitometer (Molecular Devices).

[0359] Binding of E27 variants at IgG concentration of 2 μg/mL wasexpressed as a ratio of wild type E27.

[0360] FcγRIA THP-1 Assay. 100 μL of E27 was added to the first threewells of a serocluster plate (Costar) at a concentration of 20 μg/mL inassay buffer (1×PBS, 0.1% BSA, 0.01% NaN₃). 92.5 μL of assay buffer wasadded to the next three wells followed by 7.5 μL of the 20 μg/mL E27 IgGto give a final concentration of 1.5 μg/mL. To each well, 100 μL ofTHP-1 cells were added at a concentration of 5 million cells/mL in FACSassay buffer. The plate is incubated on ice for 30 minutes

[0361] For detection, cells were washed 2×with assay buffer to removeunbound antibody. IgG binding FcγRIA was detected by adding 100 μL FITCconjugated F(ab′)₂ fragment of goat anti-human IgG heavy chain specific.(Jackson Immunoresearch) at 1:200. FITC conjugates were incubated withcells for 30 minutes on ice. Cells were washed ×3 with assay buffer toremove unbound FITC conjugate. Cells were stained with P.I. (SIGMA) at2.5 μg/mL and analyzed by flow cytometry.

[0362] Binding of E27 variants at IgG concentration of 1.5 μg/mL wasexpressed as a ratio of wild type E27.

[0363] Data from the plate assay (FcγRIA ELISA) and cell-based assay(FcγRIA THP-1 assay) was averaged to arrive at an FcγRIA-bindingactivity.

[0364] Low Affinity Fc_(γ)R Binding ELISAS: FcγRIIA, FcγRIIB andFcγRIIIA binding ELISAs were performed as described in Example 1 above,with detection of the stable hexamer (consisting of three anti-IgEmolecules and three IgE molecules).

[0365] FcRn Binding ELISA: For measuring FcRn binding activity of IgGvariants, ELISA plates were coated with 2 μg/ml streptavidin (Zymed,South San Francisco) in 50 mM carbonate buffer, pH 9.6, at 4° C.overnight and blocked with PBS-0.5% BSA, pH 7.2 at room temperature forone hour. Biotinylated FcRn (prepared using biotin-X-NHS from ResearchOrganics, Cleveland, Ohio and used at 1-2 μg/ml) in PBS-0.5% BSA, 0.05%polysorbate 20, pH 7.2, was added to the plate and incubated for onehour. Two fold serial dilutions of IgG standard (1.6-100 ng/ml) orvariants in PBS-0.5% BSA, 0.05% polysorbate 20, pH 6.0, were added tothe plate and incubated for two hours. Bound IgG was detected usingperoxidase labeled goat F(ab′)₂ anti-human IgG F(ab′)₂ in the above pH6.0 buffer (Jackson ImmunoResearch, West Grove, Pa.) followed by3,3′,5,5′-tetramethyl benzidine (Kirgaard & Perry Laboratories) as thesubstrate. Plates were washed between steps with PBS-0.05% TWEEN 20® ateither pH 7.2 or 6.0. Absorbance was read at 450 nm on a Vmax platereader (Molecular Devices, Menlo Park, Calif.). Titration curves werefit with a four-parameter nonlinear regression curve-fitting program(KaleidaGraph, Synergy software, Reading, Pa.). Concentrations of IgGvariants corresponding to the mid-point absorbance of the titrationcurve of the standard were calculated and then divided by theconcentration of the standard corresponding to the mid-point absorbanceof the standard titration curve.

[0366] In Vitro ADCC Assay: To prepare chromium 51-labeled target cells,tumor cell lines were grown in tissue culture plates and harvested usingsterile 10 mM EDTA in PBS. SK-BR-3 cells, a 3+ HER2-overexpressing humanbreast cancer cell line, were used as targets in all assays. Thedetached cells were washed twice with cell culture medium. Cells (5×10⁶)were labeled with 200 μCi of chromium51 (New England Nuclear/DuPont) at37° C. for one hour with occasional mixing. Labeled cells were washedthree times with cell culture medium, then were resuspended to aconcentration of 1×10⁵ cells/mL. Cells were used either withoutopsonization, or were opsonized prior to the assay by incubation withrhuMAb HER2 wildtype (HERCEPTIN®) or seven Fc mutants (G14, G18, G17,G36, G30, G31 and G34) at 100 ng/mL and 1.25 ng/mL in PBMC assay or 20ng/mL and 1 ng/mL in NK assay.

[0367] Peripheral blood mononuclear cells were prepared by collectingblood on heparin from normal healthy donors and dilution with an equalvolume of phosphate buffered saline (PBS). The blood was then layeredover LYMPHOCYTE SEPARATION MEDIUM® (LSM: Organon Teknika) andcentrifuged according to the manufacturer's instructions. Mononuclearcells were collected from the LSM-plasma interface and were washed threetimes with PBS. Effector cells were suspended in cell culture medium toa final concentration of 1×10⁷ cells/mL.

[0368] After purification through LSM, natural killer (NK) cells wereisolated from PBMCs by negative selection using an NK cell isolation kitand a magnetic column (Miltenyi Biotech) according to the manufacturer'sinstructions. Isolated NK cells were collected, washed and resuspendedin cell culture medium to a concentration of 2×10⁶ cells/mL. Theidentity of the NK cells was confirmed by flow cytometric analysis.

[0369] Varying effector:target ratios were prepared by serially dilutingthe effector (either PBMC or NK) cells two-fold along the rows of amicrotiter plate (100 μL final volume) in cell culture medium. Theconcentration of effector cells ranged from 1.0×10⁷/mL to 2.0×10⁴/mL forPBMC and from 2.0×10⁶/mL to 3.9×10³/mL for NK. After titration ofeffector cells, 100 μL of chromium 51-labeled target cells (opsonized ornonoponsonized) at 1×10⁵ cells/mL were added to each well of the plate.This resulted in an initial effector:target ratio of 100:1 for PBMC and20:1 for NK cells. All assays were run in duplicate, and each platecontained controls for both spontaneous lysis (no effector cells) andtotal lysis (target cells plus 100 μL) 1% sodium dodecyl sulfate, 1 Nsodium hydroxide). The plates were incubated at 37° C. for 18 hours,after which the cell culture supernatants were harvested using asupernatant collection system (Skatron Instrument, Inc.) and counted ina Minaxi auto-gamma 5000 series gamma counter (Packard) for one minute.Results were then expressed as percent cytotoxicity using the formula:

% Cytotoxicity=(sample cpm-spontaneous lysis)/(total lysis−spontaneouslysis)×100

[0370] Four-parameter curve-fitting was then used to evaluate the data(KaleidaGraph 3.0.5).

Results

[0371] A variety of antibody variants were generated which had FcRbinding activity that differed from the parent antibody. The FcR bindingdata for the variants generated is shown in Tables 6 and 7 below. Anadditional variant, T307Q, also displayed improved FcRn binding comparedto E27 parent antibody. TABLE 6 CH2 DOMAIN VARIANTS Res#EU FcRn FcγRIFcγRIIA FcγRIIB FcγRIIIA IG2 (Kabat) mean sd n mean sd n mean sd mean sdmean sd REDUCED BINDING TO ALL FcγR  1 233-236 0.54 (0.20) 3 0.12 (0.06)6 0.08 (0.01) 0.12 (0.01) 0.04 (0.02) n = 2 ELLG > PVA-  2 P238A(251)1.49 (0.17) 3 0.60 (0.05) 5 0.38 (0.14) 0.36 (0.15) 0.07 (0.05) n = 4 14D265A(278) 1.23 (0.14) 4 0.14 (0.04) 6 0.07 (0.01) 0.13 (0.05) 0.09(0.06) n = 4 17 E269A(282) 1.05 0.52 (0.03) 6 0.65 (0.18) 0.75 (0.29)0.45 (0.13) n = 5 18 D270A(283) 1.05 0.76 (0.12) 6 0.06 (0.01) 0.11(0.05) 0.14 (0.04) n = 5 58 N297A(314) 0.80 (0.18) 8 0.15 (0.06) 7 0.05(0.00) 0.10 (0.02) 0.03 (0.01) n = 3 52 A327Q(346) 0.97 0.63 (0.15) 70.13 (0.03) 0.14 (0.03) 0.06 (0.01) n = 4 64 P329A(348) 0.80 0.48 (0.10)6 0.08 (0.02) 0.12 (0.08) 0.21 (0.03) n = 4 REDUCED BINDING TO FcγRII &FcγRIII  3 S239A(252) 1.06 0.81 (0.09) 7 0.73 (0.25) 0.76 (0.36) 0.26(0.08) n = 3 33 E294A(311) 0.75 0.90 (0.08) 4 0.87 (0.19) 0.63 (0.17)0.66 (0.14) n = 5 34 Q295A(312) 0.79 1.00 (0.11) 4 0.62 (0.20) 0.50(0.24) 0.25 (0.09) n = 5 39 V303A(322) 1.26 (0.21) 3 0.91 (0.11) 5 0.86(0.10) 0.65 (0.17) 0.33 (0.09) n = 8 IMPROVED BINDING TO FcγRII &FcγRIII 11 T256A(269) 1.91 (0.43) 6 1.14 (0.14) 4 1.41 (0.27) 2.06(0.66) 1.32 (0.18) n = 9 30 K290A(307) 0.79 (0.14) 3 1.01 (0.08) 4 1.29(0.21) 1.40 (0.18) 1.28 (0.21) n = 7 44 D312A(331) 1.50 (0.06) 4 1.01(0.12) 5 1.20 (0.24) 1.19 (0.07) 1.23 (0.14) n = 3 51 K326A(345) 1.031.04 (0.05) 4 1.26 (0.21) 1.49 (0.27) 1.22 (0.28) n = 5 197  A330(349) K1.28 1.25 1.28 n = 1 273  A339(359) T 1.23 1.11 1.23 1.42 n = 1 EFFECTFcγRII 10 R255A(268) 0.59 (0.19) 4 1.26 (0.26) 8 1.30 (0.20) 1.59 (0.42)0.98 (0.18) n = 5 12 E258A(271) 1.18 1.18 (0.13) 4 1.33 (0.22) 1.65(0.38) 1.12 (0.12) n = 5 15 S267A(280) 1.08 1.20 (0.14) 4 1.64 (0.18)2.06 (0.35) 1.14 (0.25) n = 7 16 H268A(281) 1.02 (0.22) 3 1.05 (0.11) 41.22 (0.14) 1.45 (0.23) 0.52 (0.09) n = 12 19 E272A(285) 1.34 (0.24) 41.04 (0.06) 4 1.24 (0.11) 1.58 (0.19) 0.74 (0.12) n = 4 21 N276A(289)1.15 (0.21) 3 1.05 (0.14) 4 1.29 (0.20) 1.34 (0.40) 0.95 (0.04) n = 4 23D280A(295) 0.82 0.97 (0.06) 4 1.34 (0.14) 1.60 (0.31) 1.09 (0.20) n = 1025 E283A(300) 0.71 0.97 (0.03) 4 1.24 (0.23) 1.20 (0.17) 1.01 (0.14) n =5 26 H285A(302) 0.85 0.96 (0.07) 4 1.26 (0.12) 1.23 (0.15) 0.87 (0.04) n= 4 27 N286A(303) 1.24 (0.04) 2 0.94 (0.20) 13 1.28 (0.23) 1.39 (0.14)1.03 (0.08) n = 5 31 R292A(309) 0.81 (0.18) 4 0.93 (0.02) 4 0.27 (0.14)0.18 (0.07) 0.90 (0.18) n = 9 36 S298A(317) 0.80 1.10 (0.04) 3 0.40(0.08) 0.21 (0.11) 1.30 (0.18) n = 12 38 R301A(320) 0.86 1.06 (0.10) 41.12 (0.12) 1.26 (0.14) 0.21 (0.08) n = 6 38B R301M(320) 0.88 1.06(0.12) 4 1.29 (0.17) 1.56 (0.12) 0.48 (0.21) n = 4 40 V305A(324) 1.46(0.48) 6 1.04 (0.19) 10 1.12 (0.12) 1.23 (0.22) 0.84 (0.15) n = 4 41T307A(326) 1.81 (0.32) 6 0.99 (0.14) 4 1.19 (0.37) 1.35 (0.33) 1.12(0.18) n = 12 42 L309A(328) 0.63 (0.18) 4 0.93 (0.18) 6 1.13 (0.08) 1.26(0.12) 1.07 (0.20) n = 3 45 N315A(334) 0.76 (0.14) 3 1.27 (0.36) 6 1.15(0.06) 1.30 (0.17) 1.07 (0.21) n = 5 48 K320A(339) 1.10 0.98 (0.09) 51.12 (0.11) 1.22 (0.05) 0.87 (0.17) n = 4 49 K322A(341) 0.98 0.94 (0.05)6 1.15 (0.11) 1.27 (0.24) 0.61 (0.14) n = 5 50 S324A(343) 1.08 0.95(0.05) 4 0.82 (0.22) 0.70 (0.12) 1.12 (0.17) n = 4 65 P331A(350) 0.851.30 (0.34) 8 1.29 (0.14) 1.47 (0.28) 1.03 (0.19) n = 3 54 E333A(352)1.03 (0.01) 2 0.98 (0.15) 5 0.92 (0.12) 0.76 (0.11) 1.27 (0.17) n = 1056 T335A(354) 0.98 1.00 (0.05) 4 0.79 (0.22) 0.65 (0.26) 0.92 (0.54) n =3 57 S337A(356) 1.03 1.17 (0.23) 3 1.22 (0.30) 1.26 (0.06) 0.94 (0.18) n= 4 EFFECT FcγRIII  5 K248A(261) 0.87 0.95 (0.05) 5 1.06 (0.12) 1.01(0.12) 0.71 (0.05) n = 4  6 D249A(262) 0.93 1.04 (0.10) 4 1.02 (0.12)0.94 (0.02) 0.66 (0.07) n = 5  7 M252A(265) 0.64 (0.13) 4 0.99 (0.10) 51.01 (0.18) 1.15 (0.22) 0.65 (0.17) n = 6  9 S254A(267) <0.10 0.96(0.08) 4 0.97 (0.24) 1.15 (0.38) 0.73 (0.14) n = 3 16 H268A(281) 1.02(0.22) 3 1.05 (0.11) 4 1.22 (0.14) 1.45 (0.23) 0.52 (0.09) n = 12 19E272A(285) 1.34 (0.24) 4 1.04 (0.06) 4 1.24 (0.11) 1.58 (0.19) 0.74(0.12) n = 4 22 Y278A(291) 0.90 0.96 (0.02) 4 1.11 (0.08) 1.10 (0.16)0.67 (0.11) n = 4 29 T289A(306) 0.86 0.93 (0.03) 4 0.96 (0.33) 0.83(0.22) 0.62 (0.19) n = 7 32 E293A(310) 0.85 1.11 (0.07) 4 1.08 (0.19)1.07 (0.20) 0.31 (0.13) n = 6 35 Y296F(313) 0.79 1.07 (0.12) 4 0.97(0.26) 0.84 (0.18) 0.52 (0.09) n = 5 36 S298A(317) 0.80 1.10 (0.04) 30.40 (0.08) 0.21 (0.11) 1.30 (0.18) n = 12 38 R301A(320) 0.86 1.06(0.10) 4 1.12 (0.12) 1.26 (0.14) 0.21 (0.08) n = 6 38B R301M(320) 0.881.06 (0.12) 4 1.29 (0.17) 1.56 (0.12) 0.48 (0.21) n = 4 49 K322A(341)0.98 0.94 (0.05) 6 1.15 (0.11) 1.27 (0.24) 0.61 (0.14) n = 5 54E333A(352) 1.03 (0.01) 2 0.98 (0.15) 5 0.92 (0.12) 0.76 (0.11) 1.27(0.17) n = 10 55 K334A(353) 1.05 (0.03) 2 1.10 (0.06) 4 1.01 (0.15) 0.90(0.12) 1.39 (0.19) n = 17 NO EFFECT ON FcγR  4 K246A(259) 1.03 0.94(0.06) 4 1.02 (0.10) 0.92 (0.15) 1.14 (0.38) n = 4  4B K246M(259) 0.690.83 (0.05) 5 0.83 (0.06) 0.76 (0.05) 0.95 (0.09) n = 3  5B K248M(261)0.79 0.95 (0.06) 4 0.89 (0.09) 0.83 (0.04) 1.01 (0.23) n = 3  8I253A(266) <0.10 0.96 (0.05) 4 1.14 (0.02) 1.18 (0.06) 1.08 (0.14) n = 313 T260A(273) 1.09 0.93 (0.09) 4 0.89 (0.14) 0.87 (0.10) 0.89 (0.08) n =4 20 K274A(287) 1.18 1.02 (0.04) 4 0.86 (0.09) 0.96 (0.10) 1.11 (0.08) n= 3 24 V282A(299) 1.13 (0.07) 2 0.96 (0.02) 4 1.15 (0.13) 1.15 (0.20)1.00 (0.18) n = 4 28 K288A(305) 0.38 (0.12) 5 0.88 (0.15) 15 1.15 (0.26)1.14 (0.20) 1.06 (0.04) n = 4 37 Y300F(319) 0.74 (0.10) 2 1.07 (0.15) 41.11 (0.04) 1.09 (0.09) 1.01 (0.10) n = 3 43 Q311A(330) 1.62 (0.25) 40.93 (0.05) 4 1.11 (0.06) 1.19 (0.13) 0.93 (0.17) n = 3 46 K317A(336)1.44 (0.18) 4 0.92 (0.17) 6 1.13 (0.05) 1.18 (0.27) 1.10 (0.23) n = 4 47E318A(337) 0.85 0.92 (0.07) 4 1.04 (0.10) 1.17 (0.23) 1.01 (0.05) n = 353 A330Q(349) 0.76 0.96 (0.10) 4 1.01 (0.12) 1.02 (0.02) 0.75 (0.18) n =3

[0372] TABLE 7 CH3 DOMAIN VARIANTS Res#EU FcRn FcγRI FcγRIIA FcγRIIBFcγRIIIA IG2 (Kabat) mean sd n mean sd n mean sd mean sd mean sd B1K338(358)A 1.14 0.90 (0.05) 3 0.78 (0.09) 0.63 (0.08) 0.15 (0.01) n = 2B1A K338(358)M 0.78 0.99 (0.08) 3 0.99 (0.13) 0.93 (0.15) 0.49 (0.04) n= 2 B2 K340(360)A 1.02 1.04 (0.07) 3 1.05 (0.18) 0.96 (0.20) 0.84 (0.11)n = 2 B2A K340(360)M 1.20 1.17 (0.11) 3 1.10 (0.12) 1.20 (0.19) 0.75(0.12) n = 2 B3 Q342(363)A 1.09 1.13 (0.11) 3 1.01 (0.10) 1.09 (0.23)0.98 (0.10) n = 2 B4 R344(365)A 0.77 1.04 (0.08) 3 0.89 (0.14) 0.91(0.04) 0.97 (0.07) n = 4 B5 E345(366)A 1.18 1.06 (0.05) 3 1.03 (0.10)0.98 (0.10) 0.97 (0.13) n = 4 B6 Q347(368)A 0.95 1.04 (0.06) 3 1.00(0.03) 0.92 (0.02) 1.04 (0.12) n = 4 B7 R355(376)A 1.06 1.09 (0.07) 30.84 (0.09) 0.87 (0.11) 0.98 (0.09) n = 4 B8 E356(377)A 1.21 (0.11) 21.05 (0.04) 3 0.90 (0.02) 0.99 (0.13) 0.92 (0.03) n = 3 B9 M358(381)A0.96 1.06 (0.07) 3 1.11 (0.06) 1.16 (0.25) 0.91 (0.09) n = 3 B10T359(382) 1.04 1.04 (0.05) 3 1.13 (0.10) 1.15 (0.04) 1.23 (0.26) n = 3B11 K360(383)A 1.30 (0.08) 4 1.02 (0.04) 3 1.12 (0.10) 1.12 (0.08) 1.23(0.16) n = 6 B12 N361(384)A 1.16 1.00 (0.03) 3 0.82 (0.07) 0.82 (0.12)1.08 (0.06) n = 3 B13 Q362(385)A 1.25 (0.24) 3 1.00 (0.04) 3 1.03 (0.10)1.02 (0.03) 1.03 (0.16) n = 4 B14 Y373(396)A 0.86 0.98 (0.07) 3 0.84(0.11) 0.75 (0.08) 0.67 (0.04) n = 5 B15 S375(398)A 1.17 (0.19) 5 0.95(0.02) 3 1.08 (0.06) 1.14 (0.11) 1.04 (0.05) n = 6 B16 D376(399)A 1.45(0.36) 4 1.00 (0.05) 3 0.80 (0.16) 0.68 (0.14) 0.55 (0.10) n = 5 B17A378(401)Q 1.32 (0.13) 3 1.06 (0.05) 3 1.40 (0.17) 1.45 (0.17) 1.19(0.17) n = 5 B18 E380(405)A 2.19 (0.29) 6 1.04 (0.06) 3 1.18 (0.01) 1.07(0.05) 0.92 (0.12) n = 2 B19 E382(407)A 1.51 (0.18) 4 1.06 (0.03) 3 0.95(0.11) 0.84 (0.04) 0.76 (0.17) n = 3 B20 S383(408)A 0.74 1.03 (0.03) 30.92 (0.04) 0.94 (0.05) 0.88 (0.07) n = 3 B21 N384(410)A 0.88 1.00(0.01) 3 1.05 (0.19) 1.10 (0.18) 0.96 (0.18) n = 8 B22 Q386(414)A 0.70(0.10) 2 1.14 (0.08) 3 1.08 (0.13) 1.19 (0.25) 0.98 (0.14) n = 9 B23E388(416)A 0.64 (0.12) 2 1.15 (0.09) 3 0.87 (0.03) 0.94 (0.09) 0.62(0.04) n = 3 B24 N389(417)A 0.73 1.00 (0.02) 3 0.98 (0.15) 0.81 (0.04)0.75 (0.02) n = 3 B25 N390(418)A 0.87 1.06 (0.04) 3 0.99 (0.10) 0.94(0.02) 0.87 (0.09) n = 3 B26A Y391(419)A 1.14 1.00 (0.08) 3 0.97 (0.10)0.94 (0.02) 0.86 (0.05) n = 3 B26B Y391(419)F 0.81 (0.10) 2 1.00 (0.01)3 1.05 (0.12) 1.11 (0.08) 1.01 (0.15) n = 5 B27 K392(420)A 0.97 1.01(0.08) 3 0.92 (0.20) 0.94 (0.01) 0.79 (0.22) n = 3 B28 L398(426)A 0.94(0.04) 2 1.13 (0.15) 6 1.17 (0.11) 1.20 (0.08) 0.94 (0.04) n = 3 B29S400(428)A 0.64 (0.07) 3 1.10 (0.09) 3 0.95 (0.04) 0.99 (0.08) 0.83(0.07) n = 2 B30 D401(430)A 1.10 (0.09) 3 1.13 (0.16) 6 1.11 (0.12) 1.19(0.11) 0.97 (0.10) n = 5 B31 D413(444)A 1.21 (0.07) 2 1.00 (0.01) 3 0.83(0.08) 0.84 (0.06) 0.90 (0.16) n = 2 B32 K414(445)A 1.02 1.00 (0.04) 30.64 (0.15) 0.58 (0.18) 0.82 (0.27) n = 3 B33 S415(446)A 0.44 1.04(0.03) 3 0.90 (0.11) 0.88 (0.05) 0.86 (0.18) n = 2 B34 R416(447)A 1.080.96 (0.04) 3 0.68 (0.05) 0.80 (0.05) 0.71 (0.08) n = 2 B35 Q418(449)A0.77 (0.03) 2 0.98 (0.01) 3 1.00 (0.01) 0.96 (0.02) 0.96 (0.05) n = 2B36 Q419(450)A 0.76 (0.01) 2 0.97 (0.02) 3 0.68 (0.09) 0.63 (0.07) 0.86(0.08) n = 3 B37 N421(452)A 0.98 0.99 (0.01) 3 0.90 (0.03) 0.81 (0.0)0.87 (0.12) n = 2 B38 V422(453)A 1.01 0.98 (0.02) 3 0.89 (0.0) 0.83(0.05) 0.83 (0.12) n = 2 B39 S424(455)A 1.41 (0.14) 3 0.98 (0.03) 3 1.04(0.06) 1.02 (0.02) 0.88 (0.09) n = 2 B40 E430(461)A 0.93 (0.03) 2 1.05(0.02) 3 1.24 (0.11) 1.28 (0.10) 1.20 (0.18) n = 5 B41 H433(464)A 0.41(0.14) 2 0.98 (0.03) 3 0.92 (0.18) 0.79 (0.18) 1.02 (0.15) n = 3 B42N434(465)A 3.46 (0.37) 7 1.00 (0.04) 3 0.97 (0.07) 0.98 (0.13) 0.74(0.12) n = 5 B43 H435(466)A <0.10 4 1.25 (0.09) 3 0.77 (0.05) 0.72(0.05) 0.78 (0.03) n = 3 B44 Y436(467)A <0.10 2 0.99 (0.02) 2 0.93(0.05) 0.91 (0.06) 0.91 (0.15) n = 3 B45 T437(468)A 0.99 (0.07) 1.00(0.02) 3 1.12 (0.18) 1.00 (0.22) 0.77 (0.19) n = 5 B46 Q438(469)A 0.79(0.05) 2 1.02 (0.05) 3 0.80 (0.10) 0.72 (0.16) 1.01 (0.17) n = 5 B47K439(470)A 0.70 (0.04) 2 0.98 (0.04) 3 0.78 (0.16) 0.68 (0.22) 0.86(0.19) n = 4 B48 S440(471)A 0.99 1.01 (0.02) 3 1.10 (0.15) 1.11 (0.26)0.93 (0.01) n = 3 B49 S442(473)A 0.86 1.02 (0.02) 3 0.98 (0.08) 0.91(0.11) 0.95 (0.10) n = 5 B50 S444(475)A 0.80 1.01 (0.02) 3 1.07 (0.03)1.03 (0.03) 0.88 (0.12) n = 2 B51 K447(478)A 0.62 (0.12) 3 1.02 (0.03) 30.95 (0.05) 0.91 (0.05) 0.84 (0.09) n = 2

[0373] TABLE 8 NON-ALANINE VARIANTS Res#EU FcRn FcγRI FcγRIIA FcγRIIBFcγRIIIA IG2 (Kabat) mean sd n mean sd n mean sd mean sd mean sd 222D249(262)E 0.97 0.99 0.84 n = 1 176 T256(269)G 1.10 (0.03) 1.06 (0.07)0.96 (0.27) n = 2 254 T256(269)N 1.03 0.89 1.13 n = 1 157 D265(278)N0.02 (0.01) 0.03 (0.01) 0.02 (0.01) n = 3 158 D265(278)E 0.11 (0.04)0.03 (0.01) 0.02 (0.01) n = 3 189 S267(280)G R131 1.21 (0.05) 0.97(0.16) 0.09 (0.02) n = 3 H131 0.59 (0.09) n = 3 84 H268(281)N 1.33 1.410.56 n = 1 85 H268(281)S 1.35 1.38 0.81 n = 1 87 H268(281)Y 1.19 1.290.76 n = 1 168 E269(282)D 0.89 (0.10) 0.73 (0.07) 1.13 (0.21) n = 2 169E269(282)Q 0.08 (0.01) 0.16 (0.00) 0.28 (0.03) n = 2 92 D270(283)N 0.06(0.01) 0.10 (0.02) 0.04 (0.00) n = 2 93 D270(283)E 0.55 (0.05) 0.38(0.05) 1.17 (0.01) n = 2 223 E272(285)Q 1.93 1.81 0.82 n = 1 224E272(285)N 0.43 0.23 0.50 n = 1 167 K274(287)Q 0.86 0.94 0.62 n = 1 165N276(289)K 0.81 0.77 0.61 n = 1 233 N276(289)Q 1.09 0.79 0.91 n = 1 79D280(295)N 1.26 (0.07) 1.38 (0.04) 1.13 (0.13) n = 2 149 D280(295)S 1.07(0.06) 1.04 (0.08) 1.09 (0.06) n = 2 226 E283(300)Q 1.12 1.24 1.19 n = 1227 E283(300)S 1.03 1.07 0.85 n = 1 228 E283(300)N 1.18 1.28 0.94 n = 1229 E283(300)D 1.14 1.23 0.95 n = 1 23 N286(303)Q 1.52 1.13 0.96 n = 1237 N286(303)S 1.72 1.38 1.32 n = 1 238 N286(303)D 1.41 1.23 0.98 n = 173 K290(307)Q 1.17 1.26 1.40 n = 1 75 K290(307)S 1.27 1.34 1.26 n = 1 77K290(307)E 1.14 1.10 1.20 1.30 n = 1 78 K290(307)R 1.25 1.05 1.15 1.08 n= 1 177 K290(307)G 1.07 1.21 1.23 n = 1 80 R292(309)K 0.71 (0.17) 0.75(0.10) 1.15 (0.18) n = 3 81 R292(309)H 0.21 (0.09) 0.12 (0.01) 0.92(0.08) n = 2 82 R292(309)Q 0.47 (0.12) 0.25 (0.06) 0.45 (0.09) n = 3 83R292(309)N 0.54 (0.16) 0.29 (0.07) 0.88 (0.02) n = 3 144 E293(310)Q 0.85(0.03) 0.77 (0.13) 0.99 (0.04) n = 2 145 E293(310)D 0.90 (0.02) 0.88(0.07) 0.37 (0.07) n = 2 147 E293(310)K 1.13 (0.04) 1.31 (0.17) 0.72(0.08) n = 4 173 E294(311)Q 1.01 0.95 0.84 n = 1 174 E294(311)D 0.370.26 0.14 n = 1 185 Y296(313)H 0.90 0.81 0.92 n = 1 186 Y296(313)W 0.960.93 1.38 n = 1 70 S298(317)G 0.87 (0.17) 0.63 (0.33) 0.46 (0.09) n = 471 S298(317)T 0.41 (0.21) 0.40 (0.19) 0.89 (0.20) n = 3 72 S298(317)N0.08 (0.01) 0.16 (0.03) 0.06 (0.01) n = 2 218 S298(317)V 0.11 (0.06)0.17 (0.01) 0.33 (0.19) n = 3 219 S298(317)L 1.14 (0.12) 1.42 (0.31)0.34 (0.04) n = 3 150 V303(322)L 0.89 (0.05) 0.73 (0.10) 0.76 (0.09) n =4 151 V303(322)T 0.64 (0.11) 0.34 (0.05) 0.20 (0.05) n = 4 217E318(337)K 1.03 1.08 0.72 n = 1 172 K320(339)R 0.71 0.66 0.68 n = 1 202K320(339)M 1.34 1.40 1.27 n = 1 204 K320(339)Q 1.23 1.12 1.17 n = 1 205K320(339)E 1.29 1.34 1.12 n = 1 235 K320(339)R 1.24 0.95 0.86 n = 1 155K322(341)R 0.87 (0.07) 0.87 (0.21) 0.92 (0.15) n = 3 156 K322(341)Q 0.87(0.02) 0.92 (0.23) 0.78 (0.18) n = 3 206 K322(341)E 1.38 1.34 0.81 n = 1207 K322(341)N 0.57 0.36 0.04 n = 1 213 S324(343)N 1.15 1.09 0.97 n = 1214 S324(343)Q 0.82 0.83 0.78 n = 1 215 S324(343)K 0.66 0.37 0.77 n = 1216 S324(343)E 0.82 0.73 0.81 n = 1 208 K326(345)S 1.44 1.62 1.37 n = 1209 K326(345)N 1.04 1.00 1.27 n = 1 210 K326(345)Q 1.36 1.41 1.15 n = 1211 K326(345)D 1.68 2.01 1.36 n = 1 212 K326(345)E 1.34 (0.27) 1.47(0.33) 1.26 (0.04) n = 2 131 A327(346)S 0.23 (0.06) 0.22 (0.05) 0.06(0.01) n = 4 159 A327(346)G 0.92 (0.09) 0.83 (0.10) 0.36 (0.05) n = 3196 A330(349)D 0.18 0.08 0.07 n = 1 197 A330(349)K 1.28 1.25 1.28 n = 1198 P331(350)S 1.00 0.86 0.86 n = 1 199 P331(350)N 0.86 0.65 0.23 n = 1200 P331(350)E 1.06 0.91 0.42 n = 1 203 P331(350)K 0.94 0.71 0.33 n = 1141 E333(352)Q 0.70 (0.05) 0.64 (0.09) 1.10 (0.03) n = 2 142 E333(352)N0.59 (0.04) 0.52 (0.07) 0.56 (0.10) n = 2 143 E333(352)S 0.94 n = 1 152E333(352)K 0.85 (0.14) n = 3 153 E333(352)R 0.75 (0.04) 0.66 (0.03) 0.84(0.05) n = 2 154 E333(352)D 1.26 (0.04) n = 3 178 E333(352)G 0.87 0.761.05 n = 1 179 K334(353)G 0.76 (0.08) 0.60 (0.13) 0.88 (0.22) n = 5 135K334(353)R 1.15 (0.09) 1.33 (0.18) 0.68 (0.07) n = 5 136 K334(353)Q 1.08(0.11) 1.10 (0.21) 1.31 (0.26) n = 7 137 K334(353)N 1.16 (0.11) 1.29(0.30) 1.15 (0.16) n = 7 138 K334(353)S 1.01 (0.11) 1.03 ().05) 1.19(0.08) n = 3 139 K334(353)E 0.74 (0.15) 0.72 (0.12) 1.30 (0.09) n = 4140 K334(353)D 0.51 (0.09) 0.40 (0.03) 1.13 (0.09) n = 4 190 K334(353)M1.18 1.06 1.01 1.35 n = 1 191 K334(353)Y 1.15 1.08 1.05 1.31 n = 1 192K334(353)W 1.16 0.94 0.91 1.07 n = 1 193 K334(353)H 1.11 1.09 1.07 1.26n = 1 220 K334(353)V 1.13 (0.11) 1.09 (0.15) 1.34 (0.18) n = 3 221K334(353)L 1.05 1.09 1.38 n = 1 171 T335(354)Q 0.86 0.79 0.84 n = 1 194T335(354)E 1.24 1.30 1.19 n = 1 195 T335(354)K 1.19 1.14 1.30 n = 1 273A339(359)T 1.23 1.11 1.23 1.42 n = 1

[0374] The following table summarize the FcR binding activity of variouscombination variants. TABLE 9 COMBINATION VARIANTS Res#EU FcRn FcγRIFcγRIIA FcγRIIB FcγRIIIA IG2 (Kabat) mean sd n mean sd n mean sd mean sdmean sd 96 S267(280)A 1.41 1.72 0.84 n = 1 H268(281)A 134 E333(352)A0.72 (0.08) 0.63 (0.13) 1.30 (0.12) n = 5 K334(353)A 1059 T256(269)A0.44 (0.03) 0.22 (0.04) 1.41 (0.06) n = 2 S298(317)A 1051 T256(269)A0.47 (0.01) 0.30 (0.03) 1.21 (0.26) n = 2 D280(295)A S298(317)AT307(326)A 106 T256(269)A 0.11 0.08 0.90 n = 1 D280(295)A R292(309)AS298(317)A T307(326)A 107 S298(317)A 0.34 (0.05) 0.16 (0.08) 1.53 (0.24)n = 5 E333(352)A 109 S298(317)A 0.41 (0.07) 0.19 (0.08) 1.62 (0.34) n =6 K334(353)A 110 S298(317)A 0.35 (0.13) 0.18 (0.08) 1.66 (0.42) n = 11E333(352)A K334(353)A 246 S267(280)A 1.62 (0.15) 2.01 (0.45) 1.04 (0.12)n = 2 E258(271)A 247 S267(280)A 1.60 (0.18) 1.72 (0.13) 0.88 (0.07) n =3 R255(268)A 248 S267(280)A 1.54 (0.08) 1.96 (0.37) 1.13 (0.07) n = 2D280(295)A 250 S267(280)A 1.51 (0.13) 1.82 (0.32) 0.95 (0.05) n = 3E272(285)A 251 S267(280)A 1.67 (0.11) 1.85 (0.10) 0.92 (0.09) n = 3E293(310)A 264 S267(280)A 1.48 (0.12) 2.03 (0.30) 0.89 (0.04) n = 2E258(271)A D280(295)A R255(268)A 269 E380(405)A 8.55 (0.94) 3 1.02(0.07) 1.05 (0.11) 1.02 n = 2 N434(465)A 270 E380(405)A 12.6 (1.7) 0.99(0.06) 0.99 (0.11) 0.96 n = 2 N434(465)A T307(326)A 271 E380(405)A 1.01(0.01) 2 0.98 1.04 0.92 n = 1 L309(328)A 272 N434(465)A 3.15 (0.42) 20.94 (0.11) 0.96 (0.17) 0.88 n = 2 K288(305)A

Discussion

[0375] This study includes a complete mapping of human IgG1 for humanFcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and FcRn. An alanine-scan of allamino acids in human IgG1 Fc (CH2 and CH3 domains) exposed to solvent,based on the crystal structure of human Fc (Deisenhofer,Biochemistry20:2361-2370 (1981)), was performed. Each exposed amino acidin CH2 and CH3 was individually changed to alanine and the variant IgGassayed against all five human receptors; all variants were evaluatedusing humanized anti-IgE E27 IgG1 as the parent polypeptide. FcγRI andFcRn are high affinity receptors and monomeric IgG could be evaluated inthe assays for these two receptors. FcγRIIA, FcγRIIIB and FcγRIIIA arelow affinity receptors and required use of an immune complex. Hence, anELISA-type assay was used for FcγRIIA, FcγRIIB, and FcγRIIIA, in whichpre-formed hexamers, consisting of three anti-IgE E27 and three IgEmolecules were bound to the FcγR and either anti-human IgG Fc-HRP orprotein G-HRP used as detection reagent. In order to increase binding,these hexamers could be linked into multimers by addition of human VEGF(using anti-VEGF IgE). The hexamers bound to the low affinity FcγRsignificantly better than the IgG monomers; the multimers bound betterthan the hexamers (FIGS. 15A and 15B). The hexameric complexes were usedsince these provided sufficient binding and required less IgG. Complexesformed using other antibody:antigen combinations are also possiblereagents, as long as the antigen contains at least two identical bindingsites per molecule for the antibody. As an example, VEGF contains twobinding sites per VEGF dimer for anti-VEGF A.4.6.1 (Kim et al., GrowthFactors 7:53 (1992) and Kim et al. Nature 362:841 (1993)).VEGF:anti-VEGF multimers also bound to the low affinity FcγRIIA andFcγRIIIA (FIGS. 16A and 16B).

[0376] Once the complete alanine-scan was performed, several classes ofalanine variants were found. Some variants exhibited reduced binding toall FcγR (G14, FIG. 17), while other variants showed reduced bindingonly to one FcγR (G36, FIG. 17), improved binding only to one FcγR (G15,G54, G55, FIG. 17), or simultaneous reduction to one FcγR withimprovement to another (G16, FIG. 17).

[0377] Individual alanine variants were also combined in a singlevariant Fc region; e.g. combining S298(317)A with K334(353)A improvedbinding to FcγRIIIA more than either S298(317)A or K334(353)A alone(FIGS. 18A and B; and compare variants 36, 55, and 109 in Tables 6 and9) (residue numbers in parentheses are those of the EU index as inKabat). Similarly, combining S298(317)A with E333(352)A improved bindingto FcγRIIIA more than either S298(317)A or E333(352)A alone (comparevariants 36, 54, and 107 in Tables 6 and 9).

[0378] Selected IgG variants were also tested for their binding to FcγRtransfected into mammalian cells. The α-chain extracellular portion ofhuman FcγRIIIA was transfected into CHO cells using a GPI-link, whereasfor human FcγRIIB the full-length receptor was transfected into CHOcells. For the variants tested, the pattern of binding to the cells wasthe same as the pattern of binding in the protein:protein (ELISA) assay(FIGS. 18A-B and 19A-B).

[0379] One application of these variants is to improve the ADCC effectorfunction of an antibody. This can be achieved by modifying Fc regionamino acids at one or more residues which would lead to improved bindingto FcγRIIIA. Improved FcγRIIIA binding would lead to improved binding byNK cells, which carry only FcγRIIIA and can mediate ADCC. Selectedalanine variants which were either reduced in binding to FcγRIIIA(variants 17, 18, 34; Table 6), had no effect on FcγRIIIA binding(variant 31; Table 6), or had improved binding to FcγRIIIA (variants 30,36; Table 6) were tested in an in vitro ADCC assay using human PBMCs aseffector cells. Since the target cells were HER2-overexpressing SKBR3cells, the IgG Fc variants used in this assay were generated bysubstituting the V_(H)/V_(L) domains of anti-IgE E27 with those fromanti-HER2 antibody; HERCEPTIN® (humAb4D5-8 in Table 1 of Carter et al.PNAS (USA) 89:4285-4289 (1992)). The pattern of ADCC exhibited by thevariants correlated well with the pattern of binding to FcγRIIIA (FIGS.20 and 21). Notably the variant which showed the best improvement inbinding to FcγRIIIA in protein:protein assays, variant 36 S298(317)A,also showed improvement in ADCC compared to wildtype HERCEPTIN® at 1.25ng/ml (FIG. 21).

EXAMPLE 5 Bind of Fc Variants to Polymorphic Fc Receptors

[0380] Allelic variants of several of the human FcγR have been found inthe human population. These allelic variant forms have been shown toexhibit differences in binding of human and murine IgG and a number ofassociation studies have correlated clinical outcomes with the presenceof specific allelic forms (reviewed in Lehrnbecher et al. Blood94(12):4220-4232 (1999)). Several studies have investigated two forms ofFcγRIIA, R131 and H131, and their association with clinical outcomes(Hatta et al. Genes and Immunity 1:53-60 (1999); Yap et al. Lupus8:305-310 (1999); and Lorenz et al. European J. Immunogenetics22:397-401 (1995)). Two allelic forms of FcγRIIIA, F158 and V158, areonly now being investigated (Lehrnbecher et al., supra; and Wu et al. J.Clin. Invest. 100(5):1059-1070 (1997)). In this example, selected IgGvariants were tested against both allelic forms of FcγRIIA or FcγRIIIA.Fc receptor binding assays were performed essentially as described inthe above examples. However, for FcγRIIIA-V158, both (a) the lowaffinity receptor binding assay of Example 1 (which analyzes binding ofthe IgG complex to FcγRIIIA-V158); and (b) the high affinity FcγRbinding assay of Example 4 (which analyzes binding of IgG monomer toFcγRIIIA-V158) were carried out. The results of these studies aresummarized in Table 10 below. TABLE 10 Binding of Variants to FcγRIIAand FcγRIIIA Polymorphic Receptors IgG Complex IgG Complex IgG ComplexIgG Complex IgG Monomer Res#EU FcγRIIA-R131 FcγRIIA-H131 FcγRIIIA-F158FcγRIIIA-V158 FcγRIIIA-V158 IG2 (Kabat) mean sd n mean sd n mean sd nmean sd n mean sd n 11 T256(269)A 1.41 (0.27) 9 1.32 (0.18) 9 0.97(0.03) 2 1.20 1 254 T256(269)N 1.03 1 1.13 1 0.95 1 0.88 1 14 D265(278)A0.07 (0.01) 4 0.09 (0.06) 4 0.01 1 15 S267(280)A 1.64 (0.18) 7 1.05(0.03) 2 1.14 (0.25) 7 189 S267(280)G 1.21 (0.05) 3 0.59 (0.09) 3 0.09(0.02) 3 16 H268(281)A 1.22 (0.14) 12 1.09 (0.01) 2 0.52 (0.09) 12 25E283(300)A 1.24 (0.23) 5 1.01 (0.14) 5 0.78 1 226 E283(300)Q 1.12 1 1.191 0.89 1 227 E283(300)S 1.03 1 0.85 1 0.83 1 228 E283(300)N 1.18 1 0.941 0.63 1 229 E283(300)D 1.14 1 0.95 1 0.67 1 30 K290(307)A 1.29 (0.21) 71.28 (0.21) 7 1.12 (0.05) 2 1.13 1 73 K290(307)Q 1.17 1 1.40 1 1.02 11.30 1 75 K290(307)S 1.27 1 1.26 1 1.05 1 1.62 1 77 K290(307)E 1.10 11.30 1 0.98 1 1.50 1 78 K290(307)R 1.05 1 1.08 1 1.07 1 1.24 1 177K290(307)G 1.07 1 1.23 1 1.11 1 2.29 1 31 R292(309)A 0.27 (0.14) 9 0.90(0.18) 9 0.94 1 80 R292(309)K 0.71 (0.17) 3 1.15 (0.18) 3 1.64 1 81R292(309)H 0.21 (0.09) 2 0.92 (0.08) 2 1.21 1 82 R292(309)Q 0.47 (0.12)3 0.45 (0.09) 3 0.56 1 83 R292(309)N 0.54 (0.16) 3 0.88 (0.02) 3 0.91 1144 E293(310)Q 0.85 (0.03) 2 0.99 (0.04) 2 1.00 1 0.97 1 33 E294(311)A0.87 (0.19) 5 0.66 (0.14) 5 0.68 1 173 E294(311)Q 1.01 1 0.84 1 0.79 1174 E294(311)D 0.37 1 0.14 1 0.26 1 36 S298(317)A 0.40 (0.08) 12 1.30(0.18) 12 1.02 (0.04) 2 1.96 1 70 S298(317)G 0.87 (0.17) 4 0.46 (0.09) 40.88 1 1.88 1 71 S298(317)T 0.41 (0.21) 3 0.89 (0.20) 3 0.96 1 0.75 1 72S298(317)N 0.08 (0.01) 2 0.06 (0.01) 2 0.66 1 0.17 1 218 S298(317)V 0.11(0.06) 3 0.33 (0.19) 3 0.88 1 0.39 1 219 S298(317)L 1.14 (0.12) 3 0.34(0.04) 3 0.83 1 0.67 1 40 V305(324)A 1.12 (0.12) 4 1.04 1 0.84 (0.15) 441 T307(326)A 1.19 (0.37) 12 1.37 (0.13) 2 1.12 (0.18) 12 45 N315(334)A1.15 (0.06) 5 1.11 (0.06) 2 1.07 (0.21) 5 46 K317(336)A 1.13 (0.05) 41.04 1 1.10 (0.23) 4 48 K320(339)A 1.12 (0.11) 4 1.16 1 0.87 (0.17) 4 54E333(352)A 0.92 (0.12) 10 1.27 (0.17) 10 1.10 (0.10) 2 1.29 1 141E333(352)Q 0.70 (0.05) 2 1.10 (0.03) 2 1.05 1 1.00 1 142 E333(352)N 0.59(0.04) 2 0.56 (0.10) 2 0.64 1 0.56 1 143 E333(352)S 0.94 1 0.99 1 1.07 1152 E333(352)K 0.85 (0.14) 3 0.88 1 0.81 1 153 E333(352)R 0.75 (0.04) 20.84 (0.05) 2 0.93 1 0.83 1 154 E333(352)D 1.26 (0.04) 3 1.00 1 1.70 1178 E333(352)G 0.87 1 1.05 1 1.23 1 55 K334(353)A 1.01 (0.15) 17 1.39(0.19) 17 1.07 (0.09) 3 1.60 (0.01) 2 135 K334(353)R 1.15 (0.09) 5 0.68(0.07) 5 0.88 1 136 K334(353)Q 1.08 (0.11) 7 1.31 (0.26) 7 1.27 (0.01) 21.92 1 137 K334(353)N 1.16 (0.11) 7 1.15 (0.16) 7 1.19 (0.06) 2 1.70 1138 K334(353)S 1.01 (0.11) 3 1.19 (0.08) 3 1.25 1 1.82 1 139 K334(353)E0.74 (0.15) 4 1.30 (0.09) 4 1.17 1 2.75 1 140 K334(353)D 0.51 (0.09) 41.13 (0.09) 4 1.07 1 179 K334(353)G 0.76 (0.08) 5 0.88 (0.22) 5 0.94 11.28 1 190 K334(353)M 1.06 1 1.35 1 0.99 1 2.08 1 191 K334(353)Y 1.08 11.31 1 0.98 1 1.72 1 192 K334(353)W 0.94 1 1.07 1 0.96 1 1.53 1 193K334(353)H 1.09 1 1.26 1 0.97 1 2.06 1 220 K334(353)V 1.13 (0.11) 3 1.34(0.18) 3 1.00 1 2.89 1 221 K334(353)L 1.05 1 1.38 1 0.96 1 3.59 1 65P331(350)A 1.29 (0.14) 3 1.03 (0.19) 3 0.96 1 0.78 2 198 P331(350)S 1.001 0.86 1 0.54 1 199 P331(350)N 0.86 1 0.23 1 0.24 1 200 P331(350)E 1.061 0.42 1 0.36 1 203 P331(350)K 0.94 1 0.33 1 0.26 1 96 S267(280)A 1.54(0.12) 3 1.07 (0.06) 2 0.84 1 H268(281)A 110 S298(317)A 0.35 (0.13) 111.66 (0.42) 11 1.19 (0.18) 3 E333(352)A K334(353)A 271 E380(405)A 0.98 10.92 1 1.10 1 L309(328)A

[0381] For FcγRIIIA, the pattern of binding of the selected IgG1variants to the relatively higher affinity FcγRIIIA-V158 was the same asfor the relatively lower affinity FcγRIIIA-F158 (the F158 form was usedin assaying all variants). IgG1 variants which showed improved bindingto the FcγRIIIA-F158 form also showed improved binding to theFcγRIIIA-V158 form though the improvement was not as pronounced. ForFcγRIIA-R131 (used in assaying all variants) and FcγRIIA-H131, thebinding pattern of the selected IgG1 variants did show some distinctdifferences. S267(280)A, H268(281)A, and S267(280)A/H268(281)A exhibitedimproved binding to FcγRIIA-R131, compared to native IgG1, but not toFcγRIIA-H131. In contrast, S267(280)G showed improved binding toFcγRIIA-R131 but reduced binding to FcγRIIA-H131 (Table 10). Othervariants bound similarly to both allelic FcγRIIA forms: V305(324)A,T307(326)A, N315(324)A, K317(336)A, and K320(339)A.

1 11 1 218 PRT Artificial Sequence Artificial Sequence 1-218 Sequence iscompletely synthesized 1 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser LysPro Val Asp 20 25 30 Gly Glu Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln LysPro Gly 35 40 45 Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu GluSer 50 55 60 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe65 70 75 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 8085 90 Tyr Cys Gln Gln Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly 95 100105 Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe 110 115120 Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 125 130135 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 140 145150 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 155 160165 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 170 175180 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 185 190195 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 200 205210 Lys Ser Phe Asn Arg Gly Glu Cys 215 218 2 451 PRT ArtificialSequence Artificial Sequence 1-451 Sequence is completely synthesized 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr 20 25 30 SerGly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly 35 40 45 Leu GluTrp Val Ala Ser Ile Lys Tyr Ser Gly Glu Thr Lys Tyr 50 55 60 Asn Pro SerVal Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser 65 70 75 Lys Asn Thr PheTyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr TyrCys Ala Arg Gly Ser His Tyr Phe Gly His 95 100 105 Trp His Phe Ala ValTrp Gly Gln Gly Thr Leu Val Thr Val Ser 110 115 120 Ser Ala Ser Thr LysGly Pro Ser Val Phe Pro Leu Ala Pro Ser 125 130 135 Ser Lys Ser Thr SerGly Gly Thr Ala Ala Leu Gly Cys Leu Val 140 145 150 Lys Asp Tyr Phe ProGlu Pro Val Thr Val Ser Trp Asn Ser Gly 155 160 165 Ala Leu Thr Ser GlyVal His Thr Phe Pro Ala Val Leu Gln Ser 170 175 180 Ser Gly Leu Tyr SerLeu Ser Ser Val Val Thr Val Pro Ser Ser 185 190 195 Ser Leu Gly Thr GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro 200 205 210 Ser Asn Thr Lys ValAsp Lys Lys Val Glu Pro Lys Ser Cys Asp 215 220 225 Lys Thr His Thr CysPro Pro Cys Pro Ala Pro Glu Leu Leu Gly 230 235 240 Gly Pro Ser Val PheLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg ThrPro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp ProGlu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His AsnAla Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr ArgVal Val Ser Val Leu Thr Val Leu His Gln 305 310 315 Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys 320 325 330 Ala Leu Pro Ala ProIle Glu Lys Thr Ile Ser Lys Ala Lys Gly 335 340 345 Gln Pro Arg Glu ProGln Val Tyr Thr Leu Pro Pro Ser Arg Glu 350 355 360 Glu Met Thr Lys AsnGln Val Ser Leu Thr Cys Leu Val Lys Gly 365 370 375 Phe Tyr Pro Ser AspIle Ala Val Glu Trp Glu Ser Asn Gly Gln 380 385 390 Pro Glu Asn Asn TyrLys Thr Thr Pro Pro Val Leu Asp Ser Asp 395 400 405 Gly Ser Phe Phe LeuTyr Ser Lys Leu Thr Val Asp Lys Ser Arg 410 415 420 Trp Gln Gln Gly AsnVal Phe Ser Cys Ser Val Met His Glu Ala 425 430 435 Leu His Asn His TyrThr Gln Lys Ser Leu Ser Leu Ser Pro Gly 440 445 450 Lys 451 3 218 PRThomo sapiens 3 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu PhePro 1 5 10 15 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro GluVal 20 25 30 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys35 40 45 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 5055 60 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 65 7075 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 80 85 90Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 95 100 105Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 110 115 120Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 125 130 135Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 140 145 150Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 155 160 165Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 170 175 180Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 185 190 195Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 200 205 210Ser Leu Ser Leu Ser Pro Gly Lys 215 218 4 218 PRT homo sapiens 4 Pro AlaPro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 1 5 10 15 Pro LysPro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 20 25 30 Thr Cys ValVal Val Asp Val Ser His Glu Asp Pro Glu Val Lys 35 40 45 Phe Asn Trp TyrVal Asp Gly Val Glu Val His Asn Ala Lys Thr 50 55 60 Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser 65 70 75 Val Leu Thr Val Leu HisGln Asp Trp Leu Asn Gly Lys Glu Tyr 80 85 90 Lys Cys Lys Val Ser Asn LysAla Leu Pro Ala Pro Ile Glu Lys 95 100 105 Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr 110 115 120 Thr Leu Pro Pro Ser Arg AspGlu Leu Thr Lys Asn Gln Val Ser 125 130 135 Leu Thr Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val 140 145 150 Glu Trp Glu Ser Asn Gly GlnPro Glu Asn Asn Tyr Lys Thr Thr 155 160 165 Pro Pro Val Leu Asp Ser AspGly Ser Phe Phe Leu Tyr Ser Lys 170 175 180 Leu Thr Val Asp Lys Ser ArgTrp Gln Gln Gly Asn Val Phe Ser 185 190 195 Cys Ser Val Met His Glu AlaLeu His Asn His Tyr Thr Gln Lys 200 205 210 Ser Leu Ser Leu Ser Pro GlyLys 215 218 5 217 PRT homo sapiens 5 Pro Ala Pro Pro Val Ala Gly Pro SerVal Phe Leu Phe Pro Pro 1 5 10 15 Lys Pro Lys Asp Thr Leu Met Ile SerArg Thr Pro Glu Val Thr 20 25 30 Cys Val Val Val Asp Val Ser His Glu AspPro Glu Val Gln Phe 35 40 45 Asn Trp Tyr Val Asp Gly Val Glu Val His AsnAla Lys Thr Lys 50 55 60 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg ValVal Ser Val 65 70 75 Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys GluTyr Lys 80 85 90 Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu LysThr 95 100 105 Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val TyrThr 110 115 120 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val SerLeu 125 130 135 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala ValGlu 140 145 150 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr ThrPro 155 160 165 Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser LysLeu 170 175 180 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe SerCys 185 190 195 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln LysSer 200 205 210 Leu Ser Leu Ser Pro Gly Lys 215 217 6 218 PRT homosapiens 6 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 15 10 15 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 2025 30 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 35 4045 Phe Lys Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 50 55 60Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser 65 70 75 ValLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 80 85 90 Lys CysLys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 95 100 105 Thr IleSer Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 110 115 120 Thr LeuPro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 125 130 135 Leu ThrCys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 140 145 150 Glu TrpGlu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr 155 160 165 Pro ProMet Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 170 175 180 Leu ThrVal Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser 185 190 195 Cys SerVal Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys 200 205 210 Ser LeuSer Leu Ser Pro Gly Lys 215 218 7 218 PRT homo sapiens 7 Pro Ala Pro GluPhe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 1 5 10 15 Pro Lys Pro LysAsp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 20 25 30 Thr Cys Val Val ValAsp Val Ser Gln Glu Asp Pro Glu Val Gln 35 40 45 Phe Asn Trp Tyr Val AspGly Val Glu Val His Asn Ala Lys Thr 50 55 60 Lys Pro Arg Glu Glu Gln PheAsn Ser Thr Tyr Arg Val Val Ser 65 70 75 Val Leu Thr Val Leu His Gln AspTrp Leu Asn Gly Lys Glu Tyr 80 85 90 Lys Cys Lys Val Ser Asn Lys Gly LeuPro Ser Ser Ile Glu Lys 95 100 105 Thr Ile Ser Lys Ala Lys Gly Gln ProArg Glu Pro Gln Val Tyr 110 115 120 Thr Leu Pro Pro Ser Gln Glu Glu MetThr Lys Asn Gln Val Ser 125 130 135 Leu Thr Cys Leu Val Lys Gly Phe TyrPro Ser Asp Ile Ala Val 140 145 150 Glu Trp Glx Ser Asn Gly Gln Pro GluAsn Asn Tyr Lys Thr Thr 155 160 165 Pro Pro Val Leu Asp Ser Asp Gly SerPhe Phe Leu Tyr Ser Arg 170 175 180 Leu Thr Val Asp Lys Ser Arg Trp GlnGlu Gly Asn Val Phe Ser 185 190 195 Cys Ser Val Met His Glu Ala Leu HisAsn His Tyr Thr Gln Lys 200 205 210 Ser Leu Ser Leu Ser Leu Gly Lys 215218 8 215 PRT Mus musculus 8 Thr Val Pro Glu Val Ser Ser Val Phe Ile PhePro Pro Lys Pro 1 5 10 15 Lys Asp Val Leu Thr Ile Thr Leu Thr Pro LysVal Thr Cys Val 20 25 30 Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val GlnPhe Ser Trp 35 40 45 Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr GlnPro Arg 50 55 60 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu LeuPro 65 70 75 Ile Met His Gln Asp Cys Leu Asn Gly Lys Glu Phe Lys Cys Arg80 85 90 Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 95100 105 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro 110115 120 Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys 125130 135 Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln 140145 150 Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile 155160 165 Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val 170175 180 Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 185190 195 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser 200205 210 His Ser Pro Gly Lys 215 9 218 PRT Mus musculus 9 Pro Ala Pro AsnLeu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro 1 5 10 15 Pro Lys Ile LysAsp Val Leu Met Ile Ser Leu Ser Pro Ile Val 20 25 30 Thr Cys Val Val ValAsp Val Ser Glu Asp Asp Pro Asp Val Gln 35 40 45 Ile Ser Trp Phe Val AsnAsn Val Glu Val His Thr Ala Gln Thr 50 55 60 Gln Thr His Arg Glu Asp TyrAsn Ser Thr Leu Arg Val Val Ser 65 70 75 Ala Leu Pro Ile Gln His Gln AspTrp Met Ser Gly Lys Glu Phe 80 85 90 Lys Cys Lys Val Asn Asn Lys Asp LeuPro Ala Pro Ile Glu Arg 95 100 105 Thr Ile Ser Lys Pro Lys Gly Ser ValArg Ala Pro Gln Val Tyr 110 115 120 Val Leu Pro Pro Pro Glu Glu Glu MetThr Lys Lys Gln Val Thr 125 130 135 Leu Thr Cys Met Val Thr Asp Phe MetPro Glu Asp Ile Tyr Val 140 145 150 Glu Trp Thr Asn Asn Gly Lys Thr GluLeu Asn Tyr Lys Asn Thr 155 160 165 Glu Pro Val Leu Asp Ser Asp Gly SerTyr Phe Met Tyr Ser Lys 170 175 180 Leu Arg Val Glu Lys Lys Asn Trp ValGlu Arg Asn Ser Tyr Ser 185 190 195 Cys Ser Val Val His Glu Gly Leu HisAsn His His Thr Thr Lys 200 205 210 Ser Phe Ser Arg Thr Pro Gly Lys 215218 10 218 PRT Mus musculus 10 Pro Ala Pro Asn Leu Glu Gly Gly Pro SerVal Phe Ile Phe Pro 1 5 10 15 Pro Asn Ile Lys Asp Val Leu Met Ile SerLeu Thr Pro Lys Val 20 25 30 Thr Cys Val Val Val Asp Val Ser Glu Asp AspPro Asp Val Gln 35 40 45 Ile Ser Trp Phe Val Asn Asn Val Glu Val His ThrAla Gln Thr 50 55 60 Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg ValVal Ser 65 70 75 His Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys GluPhe 80 85 90 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg95 100 105 Thr Ile Ser Lys Pro Lys Gly Leu Val Arg Ala Pro Gln Val Tyr110 115 120 Thr Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser125 130 135 Leu Thr Cys Leu Val Val Gly Phe Asn Pro Gly Asp Ile Ser Val140 145 150 Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr155 160 165 Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys170 175 180 Leu Asn Met Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser185 190 195 Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys200 205 210 Thr Ile Ser Arg Ser Pro Gly Lys 215 218 11 218 PRT Musmusculus 11 Pro Pro Gly Asn Ile Leu Gly Gly Pro Ser Val Phe Ile Phe Pro1 5 10 15 Pro Lys Pro Lys Asp Ala Leu Met Ile Ser Leu Thr Pro Lys Val 2025 30 Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val His 35 4045 Val Ser Trp Phe Val Asp Asn Lys Glu Val His Thr Ala Trp Thr 50 55 60Gln Pro Arg Glu Ala Gln Tyr Asn Ser Thr Phe Arg Val Val Ser 65 70 75 AlaLeu Pro Ile Gln His Gln Asp Trp Met Arg Gly Lys Glu Phe 80 85 90 Lys CysLys Val Asn Asn Lys Ala Leu Pro Ala Pro Ile Glu Arg 95 100 105 Thr IleSer Lys Pro Lys Gly Arg Ala Gln Thr Pro Gln Val Tyr 110 115 120 Thr IlePro Pro Pro Arg Glu Gln Met Ser Lys Lys Lys Val Ser 125 130 135 Leu ThrCys Leu Val Thr Asn Phe Phe Ser Glu Ala Ile Ser Val 140 145 150 Glu TrpGlu Arg Asn Gly Glu Leu Glu Gln Asp Tyr Lys Asn Thr 155 160 165 Pro ProIle Leu Asp Ser Asp Gly Thr Tyr Phe Leu Tyr Ser Lys 170 175 180 Leu ThrVal Asp Thr Asp Ser Trp Leu Gln Gly Glu Ile Phe Thr 185 190 195 Cys SerVal Val His Glu Ala Leu His Asn His His Thr Gln Lys 200 205 210 Asn LeuSer Arg Ser Pro Gly Lys 215 218

What is claimed is:
 1. An antibody that binds IgE and comprises an Fcregion which is not a native sequence Fc region, wherein the Fc regioncomprises an amino acid substitution at amino acid position 265, whereinthe numbering of the residues in the Fc region is that of the Eu indexas in Kabat.
 2. The antibody of claim 1 wherein the amino acid residueat position 265 is alanine.
 3. A composition comprising the antibody ofclaim 1 and a pharmaceutically acceptable carrier.
 4. The composition ofclaim 3 which is sterile.
 5. An antibody variant that binds IgE andcomprises a human IgG1 Fc region, wherein the human IgG1 Fc regioncomprises an amino acid substitution at amino acid position 265, whereinthe numbering of the residues in the Fc region is that of the Eu indexas in Kabat.